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Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor
Abstract
A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegansAkt/PKB homologs, akt-1 and akt-2,transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1,revealed by a genetic screen, as well as increased dosage of wild-typeakt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 andakt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.
Keywords
• Insulin signaling
• dauer
• Fork head transcription factor
• life span
... Further studies determined loss-of-function mutation in DAF-2/IGFR can extend life span and such extension requires the IIS downstream gene daf-16 (Kenyon et al. 1993, Dorman et al. 1995, Kimura et al. 1997). In addition, loss-of-function mutations in other IIS components pdk-1, akt-1, and akt-2 are found to increase life span in a DAF-16/FoxO-dependent manner (Paradis and Ruvkun 1998, Paradis et al. 1999. ...
... Epistasis analysis revealed that loss of daf-2/IGFR promotes dauer arrest in a DAF-16/FoxO-dependent manner since daf-16(null);daf-2 double mutants do not arrest as dauers at 25°C (Riddle et al. 1981, Thomas et al. 1993, Gottlieb and Ruvkun 1994 . Similarly, daf-16/FoxO null mutation fully suppress the life span extension phenotype of daf-2 and akt-1 mutants (Kenyon et al. 1993, Paradis and Ruvkun 1998. Although DAF-16/FoxO likely regulates dauer arrest and life span via distinct mechanisms and dauer arrest is not solely regulated by IIS, dauer arrest phenotype can still provide a conveniently observable readout for assessing DAF-16/FoxO activity. ...
... ACS-3 may act on certain MUFA species to influence life span. We propose using metabolomic approach for future studies to identify the fatty acid substrates of ACS-3, which may provide mechanistic insights for the pro-longevity function of Introduction DAF-16/FoxO transcription factor is the major regulator for life span extension caused by reduced insulin/IGF-1 signaling (Kenyon et al. 1993, Paradis andRuvkun 1998). FoxO transcription factors have a conserved role in regulating life span across taxa and influence the pathogenesis of many aging-associated diseases in human (Lin et al. 1997, Bartke 2011, Shimokawa et al. 2015, Joshi et al. 2017). ...
Insulin-like signaling pathway regulates growth, reproduction, metabolism, and aging. In C. elegans, DAF-2/IGFR mutation reduces insulin-like signaling pathway activity and activates DAF-16/FoxO transcription factor to promote life span extension. We and others have shown the DAF-16A and DAF-16F isoforms are the key isoforms that control life span. To elucidate the mechanistic underpinnings of DAF-16/FoxO-dependent life span extension, we performed whole transcriptome profiling and identified isoform-specific DAF-16/FoxO target genes. acs-3, which encodes an acyl-CoA synthetase homologous to human ACSL5, was identified as a redundant target gene regulated by both DAF-16A and F. An acs-3 nonsense mutation significantly suppresses daf-2 life span extension but does not affect life span in wild-type background. To study the expression pattern of ACS-3, an ACS-3::GFP translational reporter was made and GFP expression was observed in glial, hypodermal, and vulva without obvious expression in the intestine. This ACS-3::GFP expression pattern is biologically relevant because the ACS-3::GFP transgene is able to rescue the shortened life span of acs-3;daf-2 double mutant to a comparable life span of a daf-2 single mutant. Tissue-specific experiments reconstituting ACS-3 in the tissues identified by the ACS-3::GFP translational reporter will determine the tissue requirement for rescuing the shortened life span of daf-2;acs-3 double mutant and provide insight to elucidate the molecular mechanisms of life span regulation mediated by DAF-16/FoxO. TRAP-1 (translocon-associated protein-1) is a novel regulator of insulin biogenesis in mammalian cells and specifically modulates FoxO activity through the insulin-like signaling pathway in C. elegans. Our studies found TRAP-1 is localized to the ER and loss of trap-1 activity can increase the expression level of hsp-4::GFP reporter which is a proxy for ER unfolded protein response (UPR). A genetic screen was performed to identify mutations that can modify hsp-4::GFP expression caused by trap-1 mutation (mtro screen). A col-75 missense mutant was identified as a mtro gene candidate that can increase ER UPR caused by trap-1 and col-75 single mutant is sufficient to induce hsp-4::GFP expression compared to wild-type. To validate col-75 as a bono fide mtro gene, one independent col-75 nonsense allele and two independent col-75 missense alleles were tested to determine if they are able to phenocopy our col-75 missense mutant emerged from the genetic screen. We found the nonsense allele does not induce hsp-4::GFP expression while two independent missense alleles can induce hsp-4::GFP expression stronger than wild-type, although to a lesser degree than our mutagenized allele. These results suggest our mutagenized col-75 mutant is a neomorphic allele and the increased hsp-4::GFP phenotype is not due to loss-of-function of COL-75. A col-75 transcriptional dsRed reporter strain was generated to visualize the expression pattern of COL-75 and we found col-75p::dsRed expression in the amphid socket cell, excretory cell, vulva, and phasmid sheath cell. Expression of col-75p::dsRed transgene in col-75;hsp-4::GFP background does not reveal colocalization of the two transgenes and no col-75p::dsRed signal is observed in the intestine where ER UPR is most strongly induced. These suggest col-75 can induce ER UPR cell non-autonomously. Future experiments expressing mutant collagen cDNA in the tissues identified by the col-75p::dsRed can test the ability of missense collagen variants to induce hsp-4::GFP expression cell non-autonomously and will shed light on conserved mechanisms through which collagen missense variants may influence organismal health.
... The daf-2 gene encodes the sole C. elegans homolog of IR/IGF-1R 27 . Other core components of IIS include AGE-1/PI3-K, PDK-1, AKT-1/2, and DAF-16/FoxO [27][28][29][30][31] . The IIS kinase cascade, from DAF-2 to AKT-1/2, maintains a relatively short wild-type (WT) lifespan by inhibiting the transcription factor (TF) DAF-16. ...
... Nuclear accumulation is a sign of DAF-16 activation, due to alleviation of the inhibitory phosphorylation by AKT-1/2 on DAF-16 following inactivation of AKT kinase or the upstream kinases AGE-1 and DAF-2 [27][28][29][30][31] . In adult animals, we found that endogenously expressed DAF-16::GFP from a KI allele (Fig. 2) accumulated only in the intestine following degradation of DAF-2 in the same tissue ( Fig. 4i). ...
Twenty-nine years following the breakthrough discovery that a single-gene mutation of daf-2 doubles Caenorhabditis elegans lifespan, it remains unclear where this insulin/IGF-1 receptor gene is expressed and where it acts to regulate ageing. Using knock-in fluorescent reporters, we determined that daf-2 and its downstream transcription factor daf-16 are expressed ubiquitously. Using tissue-specific targeted protein degradation, we determined that intracellular DAF-2-to-DAF-16 signaling in the intestine plays a major role in lifespan regulation, while that in the hypodermis, neurons, and germline plays a minor role. Notably, intestine-specific loss of DAF-2 activates DAF-16 in and outside the intestine, causes almost no adverse effects on development and reproduction, and extends lifespan by 94% in a way that partly requires non-intestinal DAF-16. Consistent with intestine supplying nutrients to the entire body, evidence from this and other studies suggests that altered metabolism, particularly down-regulation of protein and RNA synthesis, mediates longevity by reduction of insulin/IGF-1 signaling.
... While no differences in age-1, akt-1, and rog-1 expression were detected between animals fed the OP50 or wild type HT115 bacteria (Fig. 5A), the worms fed the mutant HT115 strain exhibited lower age-1 and akt-1 expression compared with those fed the wild type H115 (Fig. 5B). This was expected because akt-1 acts downstream of age-1 in C. elegans [42] . Interestingly, worms fed the mutant strain also showed lower expression of hlh-1 and unc54 compared with those fed the wild type strain (Fig. 5C). ...
... It has previously been reported that mutants for daf-2 (insulin/IGF receptor) and age-1 (catalytic subunit of phosphatidylinositol 3-OH kinase) exhibit enhanced resistance to PA14 infection (41), similarly to inx-14 mutants described here. When, we performed daf-2 or age-1 RNAi knockdown in inx-14(ag17) mutants, we observed a significantly stronger resistance to PA14 than in either single mutant (Figures 3D, E), while daf-16 RNAi in wild-type or inx-14(ag17) mutant animal did not alter the sensitivity of these strains ( Figure 3F). ...
Gap junctions mediate intercellular communications across cellular networks in the nervous and immune systems. Yet their roles in intestinal innate immunity are poorly understood. Here, we show that the gap junction/innexin subunit inx-14 acts in the C. elegans gonad to attenuate intestinal defenses to Pseudomonas aeruginosa PA14 infection through the PMK-1/p38 pathway. RNA-Seq analyses revealed that germline-specific inx-14 RNAi downregulated Notch/GLP-1 signaling, while lysosome and PMK-1/p38 pathways were upregulated. Consistently, disruption of inx-14 or glp-1 in the germline enhanced resistance to PA14 infection and upregulated lysosome and PMK-1/p38 activity. We show that lysosome signaling functions downstream of the INX-14/GLP-1 signaling axis and upstream of PMK-1/p38 pathway to facilitate intestinal defense. Our findings expand the understanding of the links between the reproductive system and intestinal defense, which may be evolutionarily conserved in higher organism.
... The canonical C. elegans IIS pathway begins with insulin-like ligands, which bind and activate or inhibit DAF-2. Receptor activation recruits phosphoinositide-3 kinase, AGE-1/PI3K, which initiates a signaling cascade that includes PIP 3 , and the serine/ threonine kinases PDK-1, AKT-1, and AKT-2 (Dorman et al., 1995;Morris et al., 1996;Paradis and Ruvkun, 1998;Paradis et al., 1999). Then, transcription factors are inhibited, including DAF-16/FOXO and SKN-1/Nrf2, preventing activation of downstream target genes that promote longevity. ...
The Transforming Growth Factor-β (TGF-β) superfamily of signaling molecules plays critical roles in development, differentiation, homeostasis, and disease. Due to the conservation of these ligands and their signaling pathways, genetic studies in invertebrate systems including the nematode Caenorhabditis elegans have been instrumental in identifying signaling mechanisms. C. elegans is also a premier organism for research in longevity and healthy aging. Here we summarize current knowledge on the roles of TGF-β signaling in aging and immunity.
... The lower number of pachytene stage nuclei in daf-2 may not be simply due to increased apoptosis as daf-16;daf-2 also showed similar elevated numbers ( Figure S2G). We also discovered that components of the canonical IIS signaling pathway are involved, as cdk-12 KD in age-1(hx546) (mammalian PI3K ortholog) (Morris et al., 1996;Paradis and Ruvkun, 1998) and pdk-1(sa680) (mammalian PDK ortholog) (Paradis et al., 1999) also arrested germ line at the pachytene stage of meiosis ( Figure S2H, I), similar to daf-2 on cdk-12 RNAi. ...
Germ line integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signaling to protect the germ line from genotoxic stress, facilitating the cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germ line quality in response to DNA damage is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic D NA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signaling (IIS) is low, arrests oogenesis in the pachytene stage of meiosis I, in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape the arrest to produce poor-quality oocytes, showing that the TF imposes strict quality control during low IIS. Activated FOXO/DAF-16 senses DDR perturbations during low IIS to lower ERK/MPK-1 signaling below a threshold to promote germ line arrest. Altogether, we elucidate a new surveillance role of activated FOXO/DAF-16 that ensures optimal germ cell quality and progeny fitness in response to somatic DNA damage.
... To investigate organism-wide DAF-16/FOXO dynamics, we studied the C. elegans L1 arrest, a developmental arrest larvae enter when they encounter high stresses, including starvation, osmotic shock and heat, directly after hatching and that is under control of IIS [20,[22][23][24]28,29] (Fig. 1A). Under unstressed conditions, high insulin-like peptide (ILP) levels activate the insulin receptor DAF-2, causing phosphorylation and cytoplasmic localization of DAF-16/FOXO [30][31][32]. Upon stress, low ILP levels result in DAF-2 inactivation, DAF-16/FOXO dephosphorylation and its subsequent translocation into in the nucleus, where it induces stress response genes [33][34][35] and regulators of cell proliferation [22], ultimately leading to developmental arrest. ...
FOXO transcription factors are highly conserved effectors of insulin and insulin-like growth factor signaling, that are crucial for mounting responses to a broad range of stresses. Key signaling step is the stress-induced translocation of FOXO proteins to the nucleus, where they induce expression of stress response genes. Insulin signaling and FOXO proteins often control responses that impact the entire organism, such as growth or starvation-induced developmental arrest, but how body-wide coordination is achieved is poorly understood. Here, we leverage the small size of the nematode C. elegans, to quantify translocation dynamics of DAF-16, the sole C. elegans FOXO transcription factor, with single-cell resolution, yet in a body-wide manner. Surprisingly, when we exposed individual animals to constant levels of stress that cause larval developmental arrest, DAF-16/FOXO translocated between the nucleus and cytoplasm in stochastic pulses. Even though the occurrence of translocation pulses was random, they nevertheless exhibited striking synchronization between cells throughout the body. DAF-16/FOXO pulse dynamics were strongly linked to body-wide growth, with isolated translocation pulses causing transient reduction of growth and full growth arrest observed only when pulses were of sufficiently high frequency or duration. Finally, we observed translocation pulses of FOXO3A in mammalian cells under nutrient stress. The link between DAF-16/FOXO pulses and growth provides a rationale for their synchrony, as uniform proportions are only maintained when growth and, hence, pulse dynamics are tightly coordinated between all cells. Long-range synchronization of FOXO translocation dynamics might therefore be integral also to growth control in more complex animals.
... Insulin receptor substrate 1 (IRS-1) and insulin receptor substrate 2 (IRS-2), the dominant substrate proteins of the insulin receptor, are linked to the activation of phosphatidylinositol 3-kinase (PI3K) [5,6]. This enzyme in combination with dehydrogenase kinase isozyme 1 (PDK1) activates Akt through phosphorylation [7]. Following the pathway, the cytoplasmic glycogen synthase kinase-3β (GSK-3β) is deactivated by the Akt via phosphorylation [8]. ...
Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities. In this study, apigenin, luteolin, and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant (IR) HepG2 cells. All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4 (GLUT4) and phosphor-glycogen synthase kinase (GSK-3β). These fl avonoids signifi cantly inhibited the production of reactive oxygen species (ROS) and advanced glycation end-products (AGEs), which were closely related to the suppression of the phosphorylation form of NF-κB and P65. The expression levels of insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells were all partially activated by the fl avonoids, with variable effects. Furthermore, the intracellular metabolic conditions of the fl avonoids were also evaluated.
... In the IIS pathway, the agonist insulin-like peptide binds to the receptor daf-2 and recruits the insulin receptor substrate, leading to activation of age-1 and akt-1 . Phosphorylation that activates the downstream kinase cascade causes inactivation of the DAF-16/FoxO transcription factor, which hinders lifespan (Paradis et al. 1998). Exposure to environmental pressures like oxidative stress or heat shock can negatively regulate IIS and activate DAF-16, which induces the transcription of downstream effector genes, thus prolonging longevity (Meng et al. 2018). ...
Phlorizin, a natural compound, was studied using C. elegans to explore its potential mechanism for extending lifespan. Results showed that phlorizin mitigated the harmful effects of high temperatures and hydrogen peroxide, reduced oxidative stress, increased antioxidant enzyme activity, and reduced MDA levels. Through network pharmacological analysis, it was determined that the AKT1, INSR and SOD2 signaling pathway plays a key role in the anti-aging effects of phlorizin. Experiments with mutants and transgenic C. elegans showed that phlorizin lifespan extension and antioxidant effects are mainly mediated by insulin and SIR-2.1 pathways that regulate DAF-16 and SKN-1 nuclear translocation and its downstream targets SOD-3 and HSP-16.2. These findings were supported by molecular docking experiments. Overall, this study provides insight into the potential use of phlorizin as an anti-aging agent and highlights the importance of the DAF-16/SOD-3 signaling pathway in mediating its action.
... Savory et al. also measured the lifespan of animals grown to adulthood at 20 • C and then transferred to 4 • C and found that AGE-1 negatively regulates survival at 4 • C (Savory et al., 2011). AGE-1 is a phosphatidylinositol 3-kinase that functions downstream of the DAF-2 receptor ( Fig. 2) (Paradis and Ruvkun, 1998). This supports our hypothesis that the insulin pathway is activated during high-temperature cultivation. ...
Acclimation to temperature is one of the survival strategies used by organisms to adapt to changing environmental temperatures. Caenorhabditis elegans' cold tolerance is altered by previous cultivation temperature, and similarly, past low-temperature induces a longer lifespan. Temperature is thought to cause a large shift in homeostasis, lipid metabolism, and reproduction in the organism because it is a direct physiological factor during chemical events. This paper will share and discuss what we know so far about the neural and molecular mechanisms that control cold tolerance and lifespan by altering lipid metabolism and physiological characteristics. We hope that this will contribute to a better understanding of how organisms respond to temperature changes.
... The insulin signaling propagates from AGE-1/PI3K phosphorylating the AKT-1/2 and activating the PDK-1 kinase to retain the transcription factors DAF-16/FOXO and SKN-1/Nrf1,2,3 in the cytoplasm [46]. Upon reduction of DAF-2/Insulin/IGF-1 receptor signaling, DAF-16/FOXO and SKN-1/Nrf1,2,3 translocate to the nucleus and activate a number of protective genes [47][48][49]. Furthermore, mutations in the homologous insulin signaling cascade in Drosophila melanogaster (D. melanogaster) have also shown significant increases in lifespan [50]. ...
The biomechanical properties of extracellular matrices (ECM) and their consequences for cellular homeostasis have recently emerged as a driver of aging. Here we review the age-dependent deterioration of ECM in the context of our current understanding of the aging processes. We discuss the reciprocal interactions of longevity interventions with ECM remodeling. And the relevance of ECM dynamics captured by the matrisome and the matreotypes associated with health, disease, and longevity. Furthermore, we highlight that many established longevity compounds promote ECM homeostasis. A large body of evidence for the ECM to qualify as a hallmark of aging is emerging, and the data in invertebrates is promising. However, direct experimental proof that activating ECM homeostasis is sufficient to slow aging in mammals is lacking. We conclude that further research is required and anticipate that a conceptual framework for ECM biomechanics and homeostasis will provide new strategies to promote health during aging.
... 32,36 PIP 3 , in turn, activates the 3-phosphoinositide-dependent protein kinase 1 (PDK-1), which then phosphorylates and activates protein kinases B (AKT1−2) and serum-and glucocorticoid-regulated kinase 1 (SGK-1). 37,38 These molecules phosphorylate and inactivate DAF-16 (FOXO transcription factor homologous), SKN-1 (Nrf2 homologous), and heat shock transcription factor 1 (HSF-1), impeding their translocation to the nucleus. Nevertheless, under unfavorable conditions, such as oxidative stress, IIS is hampered by DAF-18 (PTEN homologous). ...
Oxidative stress is a natural physiological process where the levels of oxidants, such as reactive oxygen species (ROS) and nitrogen (RNS), exceed the strategy of antioxidant defenses, culminating in the interruption of redox signaling and control. Oxidative stress is associated with multiple pathologies, including premature aging, neurodegenerative diseases, obesity, diabetes, atherosclerosis, and arthritis. It is not yet clear whether oxidative stress is the cause or consequence of these diseases; however, it has been shown that using compounds with antioxidant properties, particularly compounds of natural origin, could prevent or slow down the progress of different pathologies. Within this context, the Caenorhabditis elegans (C. elegans) model has served to study the effect of different metabolites and natural compounds, which has helped to decipher molecular targets and the effect of these compounds on premature aging and some diseases such as neurodegenerative diseases and dyslipidemia. This article lists the studies carried out on C. elegans in which metabolites and natural extracts have been tested against oxidative stress and the pathologies associated with providing an overview of the discoveries in the redox area made with this nematode.
... The global proteomic analysis also revealed some common changes in all strains following exercise, AKT-1 and DUF148 increased in abundance. AKT-1 is involved in the phosphorylation of DAF-16 and SKN-1, inhibiting their translocation to the nucleus [40,51]. Similarly, HACD-1 and PMT-1 decreased in abundance following exercise in the 3 strains. ...
Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle that promotes changes in gene transcription and mitochondrial biogenesis, required for the beneficial adaptive response. We demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive hormesis response to physiological levels of H2O2 in myoblasts and following exercise in C. elegans. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. Moreover, a swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of specific Cysteine residues revealed a more reduced redox state in the non-exercised prdx-2 mutant strain that become oxidized following exercise. In contrast, specific Cys residues from regulatory proteins become more reduced in the N2 strain following exercise, establishing the key regulatory role of PRDX-2 in a redox signalling cascade following endogenous ROS generation. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological redox stress.
... The researches show that local inhibition of signal pathway mediated by insulin like growth factor (Insulin-like growth factors, IGFs) lead to an increase in invertebrate and vertebrate model organisms, which may delay the aging [1]. The study of Caenorhabditis elegans for the first time found that the inhibition of signaling pathway of DAF-2 (encoding insulin-like growth factor-1 receptor,IGF-1R) could increase the lifespan [2][3][4]. By target mutation, the gene study encoding the invertebrate life has made significant progress, and the research elucidates the inhibition of signal pathway of insulin or insulin-like growth factor-1 can significantly increase the lifespan [5] In fruit flies, yeast and mammals, insulin like signaling cascade, similarly to the DAF-2 signaling pathway, can increase the lifespan by altering the IGF-1 signal pathway. ...
... We combined literature-reported regulations into a preliminary model and used results obtained from simulations and perturbations to fine-tune the Boolean functions for each node until we were able to mimic the expected behavior of the system. For example, multiple articles reported that daf-16 was activated either when akt was repressed (Henderson & Johnson, 2001;Lin et al., 2001;Lee, Hench & Ruvkun, 2001;Paradis & Ruvkun, 1998) or when daf-12 was activated (Matyash et al., 2004;Dowell et al., 2003). Simulated conditions for normal adult development should therefore result in daf-16 repression and no dauer activation. ...
Cellular fates are determined by genes interacting across large, complex biological networks. A critical question is how to identify causal relationships spanning distinct signaling pathways and underlying organismal phenotypes. Here, we address this question by constructing a Boolean model of a well-studied developmental network and analyzing information flows through the system. Depending on environmental signals Caenorhabditis elegans develop normally to sexual maturity or enter a reproductively delayed, developmentally quiescent ‘dauer’ state, progressing to maturity when the environment changes. The developmental network that starts with environmental signal and ends in the dauer/no dauer fate involves genes across 4 signaling pathways including cyclic GMP, Insulin/IGF-1, TGF- β and steroid hormone synthesis. We identified three stable motifs leading to normal development, each composed of genes interacting across the Insulin/IGF-1, TGF- β and steroid hormone synthesis pathways. Three genes known to influence dauer fate, daf-2 , daf-7 and hsf-1 , acted as driver nodes in the system. Using causal logic analysis, we identified a five gene cyclic subgraph integrating the information flow from environmental signal to dauer fate. Perturbation analysis showed that a multifactorial insulin profile determined the stable motifs the system entered and interacted with daf-12 as the switchpoint driving the dauer/no dauer fate. Our results show that complex organismal systems can be distilled into abstract representations that permit full characterization of the causal relationships driving developmental fates. Analyzing organismal systems from this perspective of logic and function has important implications for studies examining the evolution and conservation of signaling pathways.
... The global proteomic analysis also revealed some common changes in all strains following exercise, AKT-1 and DUF148 12 increased in abundance. AKT-1 is involved in the phosphorylation of DAF-16 and SKN-1, inhibiting their translocation to the nucleus [39,48]. Similarly, HACD-1 and PMT-1 decreased 14 in abundance following exercise in the 3 strains. ...
Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle required for a beneficial adaptive response by activation of specific signalling pathways. Here, we demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive beneficial hormesis response to H2O2. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. A swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of individual Cysteine residues revealed a relatively more oxidised redox state following exercise in the prdx-2 mutants. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological stress.
... Moreover, recent findings suggest that maintenance of proteostasis may in part underlie FOXO's role as a pro-longevity factor. In C. elegans, FoxO activation is concurrent with hypometabolism, and an absence of Akt signaling leads to activation of the FoxO homologue, DAF-16, and entrance into the quiescent dauer state (Ogg et al. 1997;Paradis & Ruvkun 1998). In addition, studies in mammalian cells have shown that activation of FoxO proteins is associated with survival under fasting conditions by facilitating cell cycle arrest (van der Horst & Burgering 2007). ...
... In C. elegans, mutation of Daf-16 (C. elegans FoxO) can affect dauer formation and shorten the lifespan (25). In Drosophila melanogaster, knockout of FoxO shortens the lifespan, and activation of FoxO is a well-established lifespan-extending condition (26). ...
Diapause is a form of dormancy used widely by insects to survive adverse seasons. Previous studies have demonstrated that forkhead box O (FoxO) is activated during pupal diapause initiation in the moth Helicoverpa armigera. However, it is unclear how FoxO induces diapause. Here, we show that knockout of FoxO causes H. armigera diapause-destined pupae to channel into nondiapause, indicating that FoxO is a master regulator that induces insect diapause. FoxO activates the ubiquitin-proteasome system (UPS) by promoting ubiquitin c (Ubc) expression via directly binding to the Ubc promoter. Activated UPS decreases transforming growth factor beta (TGFβ) receptor signaling via ubiquitination to block developmental signaling to induce diapause. This study significantly advances the understanding of insect diapause by uncovering the detailed molecular mechanism of FoxO.
... This initiates an intracellular signaling cascade of protein kinases: AAP-1, the p55-like regulatory subunit of PI3K, is activated which enables the conversion of PIP2 to PIP3 by AGE-1, the catalytic subunit of PI3K (Morris et al., 1996;Wolkow et al., 2000). PDK-1 transduces the phosphorylation signal from PIP3 to SGK-1, AKT-1 and AKT-2 (Hertweck et al., 2004;Paradis et al., 1999;Paradis & Ruvkun, 1998) which in turn inactivates the FoxO transcription factor DAF-16 (Lin et al., 1997;Ogg et al., 1997). When DAF-16 localizes to the nucleus, an enhanced life maintenance program is activated that increases lifespan (Henderson & Johnson, 2001;Lin et al., 2001)(figure 1.7). ...
... IIS is also evolutionarily conserved in Drosophila and mice, and probably, in humans (Fontana et al., 2010). In this pathway, the ligand binding to DAF-2/InR activates its tyrosine kinase activity and initiates a cascade of sequential phosphorylation of kinases: AGE-1/PI3K, PDK-1, AKT-1/2, and SGK-1 (Paradis and Ruvkun, 1998). Then, AKT and SGK-1 phosphorylate and inactivate the FOXO transcription factor DAF-16 by preventing its translocation to the nucleus. ...
Aging is associated with the increased risk of most age-related diseases in humans. Complanatoside A (CA) is a flavonoid compound isolated from the herbal medicine Semen Astragali Complanati. CA was reported to have potential anti-inflammatory and anti-oxidative activities. In this study, we investigated whether CA could increase the stress resistance capability and life span of Caenorhabditis elegans. Our results showed that CA could extend the longevity of C. elegans in a dosage-dependent manner, while 50 μM of CA has the best effect and increased the life span of C. elegans by about 16.87%. CA also improved the physiological functions in aging worms, such as enhanced locomotor capacity, and reduced the accumulation of the aging pigment. CA could also reduce the accumulation of toxic proteins (α-synuclein and β-amyloid) and delay the onset of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, in models of C. elegans. Further investigation has revealed that CA requires DAF-16/FOXO, SKN-1, and HSF-1 to extend the life span of C. elegans. CA could increase the antioxidation and detoxification activities regulated by transcription factor SKN-1 and the heat resistance by activating HSF-1 that mediated the expression of the chaperone heat shock proteins. Our results suggest that CA is a potential antiaging agent worth further research for its pharmacological mechanism and development for pharmaceutical applications.
... C. elegans AKT-1 and AKT-2 function upstream of, and can phosphorylate, DAF-16 [43,44]. The SGK-1 Serum Glucorticoid Kinase homolog interacts with AKT kinases and has been shown to regulate DAF-16 nuclear localization [44]. ...
FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging.
... Accumulation of PIP 3 activates PDK-1 (PDPK1, 3phosphoinositide-dependent protein kinase-1), which activates AKT-1/2 (AKT Serine/Threonine Kinase, protein kinase B) via phosphorylation (Paradis et al., 1999). In turn activated AKT-1/2 directly inhibits DAF-16 (FOXO) through phosphorylation and sequestration in the cytosol (Paradis and Ruvkun 1998;Lee et al., 2001;Lin et al., 2001). Mutations within this pathway that decreased ILS lead to activation of DAF-16 and increased longevity (Kenyon et al., 1993;Lin et al., 1997;Ogg et al., 1997). ...
Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.
... Conversely, in the insulin/IGF pathway, the DAF-2 insulin/IGF receptor transmits the signal through a cascade of phosphorylation events, which culminates in regulation of the FOXO transcription factor DAF-16 ( Fig. 10.2) (Gottlieb & Ruvkun, 1994;Kimura et al., 1997;Ogg et al., 1997;Ogg & Ruvkun, 1998;Paradis & Ruvkun, 1998;Paradis et al., 1999;Lee et al., 2001;Wolkow et al., 2002;Li et al., 2003). Downregulation of insulin signalling and downstream kinases leads to nuclear translocation of unphosphorylated DAF-16, which is required for dauer formation (Lee et al., 2001;Lin et al., 2001;Hertweck et al., 2004). ...
... Next, we tested whether overactivation of the PI3K pathway could rescue the invasion defects caused by down-regulation of mcm-7. For this purpose, we performed mcm-7 RNAi in animals carrying a gain-of-function mutation in the AKT homolog akt-1 [43] or a deletion allele of daf-18, which encodes the homolog of the PTEN tumor suppressor that antagonizes PI3K activity [42], and scored BM breaching using the LAM-1::GFP marker. The akt-1 (mg144gf) and daf-18(ok480lf) mutations partially suppressed the invasion defects caused by mcm-7 RNAi, while the egl-43 RNAi invasion defects were not affected by akt-1(gf) or daf-18 (lf) (Fig 5H). ...
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
... Muscle hypertrophy can be induced by Gh, resulting in increased myoblast fusion and myofibre size [115], and IGF-1, which results in increased protein synthesis and a downregulation of muscle-specific ubiquitin ligases [37]. The relationship of IGF and its downstream pathway in muscle growth or degradation has been determined in mice [78,84,106], fish [12,20,34], chicken [86], and the invertebrate models fruit fly [36,124], nematode [94], oyster [51] and hydroid [72] responding to nutritional state. ...
Background
The formation and functioning of muscles are fundamental aspects of animal biology, and the evolution of ‘muscle genes’ is central to our understanding of this tissue. Feeding-fasting-refeeding experiments have been widely used to assess muscle cellular and metabolic responses to nutrition. Though these studies have focused on vertebrate models and only a few invertebrate systems, they have found similar processes are involved in muscle degradation and maintenance. Motivation for these studies stems from interest in diseases whose pathologies involve muscle atrophy, a symptom also triggered by fasting, as well as commercial interest in the muscle mass of animals kept for consumption. Experimentally modelling atrophy by manipulating nutritional state causes muscle mass to be depleted during starvation and replenished with refeeding so that the genetic mechanisms controlling muscle growth and degradation can be understood.
Results
Using amphioxus, the earliest branching chordate lineage, we address the gap in previous work stemming from comparisons between distantly related vertebrate and invertebrate models. Our amphioxus feeding-fasting-refeeding muscle transcriptomes reveal a highly conserved myogenic program and that the pro-orthologues of many vertebrate myoblast fusion genes were present in the ancestral chordate, despite these invertebrate chordates having unfused mononucleate myocytes. We found that genes differentially expressed between fed and fasted amphioxus were orthologous to the genes that respond to nutritional state in vertebrates. This response is driven in a large part by the highly conserved IGF/Akt/FOXO pathway, where depleted nutrient levels result in activation of FOXO, a transcription factor with many autophagy-related gene targets.
Conclusion
Reconstruction of these gene networks and pathways in amphioxus muscle provides a key point of comparison between the distantly related groups assessed thus far, significantly refining the reconstruction of the ancestral state for chordate myoblast fusion genes and identifying the extensive role of duplicated genes in the IGF/Akt/FOXO pathway across animals. Our study elucidates the evolutionary trajectory of muscle genes as they relate to the increased complexity of vertebrate muscles and muscle development.
... This association was first identified in C. elegans, in which the loss of FOXO family members rescued dauer-stage arrest caused by the depletion of Akt-1 and Akt-2. 98 The activity and substrates of Akt are regulated by the phosphorylation of threonine 308 and serine 473, 99 and the phosphorylation of serine 473 is not essential for the Akt-mediated phosphorylation of TSC2 and GSK3β; however, it is essential for the phosphorylation and inactivation of FOXOs. 100 In summary, these biochemical and genetic studies have confirmed that the main phenotypes induced by Akt depletion are driven by FOXO-mediated transcription; therefore, FOXOs are downstream targets of Akt signaling in various biological reactions. The activation of Akt signaling can promote (arrows) or inhibit (blocking arrows) the phosphorylation of downstream effectors. ...
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a crucial role in various cellular processes and is aberrantly activated in cancers, contributing to the occurrence and progression of tumors. Examining the upstream and downstream nodes of this pathway could allow full elucidation of its function. Based on accumulating evidence, strategies targeting major components of the pathway might provide new insights for cancer drug discovery. Researchers have explored the use of some inhibitors targeting this pathway to block survival pathways. However, because oncogenic PI3K pathway activation occurs through various mechanisms, the clinical efficacies of these inhibitors are limited. Moreover, pathway activation is accompanied by the development of therapeutic resistance. Therefore, strategies involving pathway inhibitors and other cancer treatments in combination might solve the therapeutic dilemma. In this review, we discuss the roles of the PI3K/Akt pathway in various cancer phenotypes, review the current statuses of different PI3K/Akt inhibitors, and introduce combination therapies consisting of signaling inhibitors and conventional cancer therapies. The information presented herein suggests that cascading inhibitors of the PI3K/Akt signaling pathway, either alone or in combination with other therapies, are the most effective treatment strategy for cancer.
... The canonical insulin and growth factor signaling initiates when secreted insulin or insulin-like growth factors (IGFs) bind to their cell surface receptors. Dimerized receptors trigger a series of autophosphorylation and recruit insulin receptor substrate 1-4 (IRS1-4) and phosphatidylinositol 3-kinase (PI3K), the latter increases the local concentrations of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 acts as a second messenger to activate phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (AKT or PKB). ...
Aging happens to all of us as we live. Thanks to the improved living standard and discovery of life-saving medicines, our life expectancy has increased substantially across the world in the past century. However, the rise in lifespan leads to unprecedented increases in both the number and the percentage of individuals 65 years and older, accompanied by the increased incidences of age-related diseases such as type 2 diabetes mellitus and Alzheimer’s disease. FoxO transcription factors are evolutionarily conserved molecules that play critical roles in diverse biological processes, in particular aging and metabolism. Their dysfunction is often found in the pathogenesis of many age-related diseases. Here, we summarize the signaling pathways and cellular functions of FoxO proteins. We also review the complex role of FoxO in aging and age-related diseases, with focus on type 2 diabetes and Alzheimer’s disease and discuss the possibility of FoxO as a molecular link between aging and disease risks.
... Another 12 INSs have an antagonistic effect on DAF-2, promoting dauer [59]. Through DAF-2, the phosphoinositide-3-kinase (PI3K) AGEing alteration 1 (AGE-1) is activated, and this phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP 2 ) into phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) in turn activates downstream kinases PDK-1, AKT-1 and AKT-2 [60][61][62]. AGE-1 is antagonized by DAF-18/PTEN [34]. AKT-1/2 phosphorylate the FoxO transcription factor DAF-16, thereby sequestering it in the cytoplasm (ref. ...
Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8–12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans , and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed.
... Insulin-response sequence mediates the Akt-dependent transcriptional repression of these genes [203]. In some studies, it was observed that inhibition of DAF-16 (Forkhead family member) by PI3K-Akt pathway through phosphorylation is the major target [204]. There are three representatives of the Forkhead family viz. ...
Alzheimer’s disease (AD) is the foremost type of dementia that afflicts considerable morbidity and mortality in aged population. Several transcription molecules, pathways, and molecular mechanisms such as oxidative stress, inflammation, autophagy, and immune system interact in a multifaceted way that disrupt physiological processes (cell growth, differentiation, survival, lipid and energy metabolism, endocytosis) leading to apoptosis, tauopathy, β-amyloidopathy, neuron, and synapse loss, which play an important role in AD pathophysiology. Despite of stupendous advancements in pathogenic mechanisms, treatment of AD is still a nightmare in the field of medicine. There is compelling urgency to find not only symptomatic but effective disease-modifying therapies. Recently, phosphoinositide 3-kinase (PI3K) and Akt are identified as a pathway triggered by diverse stimuli, including insulin, growth factors, cytokines, and cellular stress, that link amyloid-β, neurofibrillary tangles, and brain atrophy. The present review aims to explore and analyze the role of PI3K-Akt pathway in AD and agents which may modulate Akt and have therapeutic prospects in AD. The literature was researched using keywords “PI3K-Akt” and “Alzheimer’s disease” from PubMed, Web of Science, Bentham, Science Direct, Springer Nature, Scopus, and Google Scholar databases including books. Articles published from 1992 to 2021 were prioritized and analyzed for their strengths and limitations, and most appropriate ones were selected for the purpose of review. PI3K-Akt pathway regulates various biological processes such as cell proliferation, motility, growth, survival, and metabolic functions, and inhibits many neurotoxic mechanisms. Furthermore, experimental data indicate that PI3K-Akt signaling might be an important therapeutic target in treatment of AD.
... DBL-1, the C. elegans BMP2/4 homolog, plays a major role in body size regulation, male tail development, mesodermal patterning, and lipid accumulation [1,11,17]. The IIS pathway in C. elegans uses a single insulin receptor DAF-2/InsR [18,19] in conjunction with 40 insulin-like peptides (ILPs) to regulate multiple homeostatic functions through the control of transcription factors, such as DAF-16/FoxO [20][21][22] and SKN-1/ Nrf [23]. DAF-2/InsR has prolific effects on the development and homeostasis of the worm; disruptions to DAF-2/InsR lead to phenotypes in dauer formation, longevity, stress tolerance, innate immunity, germline maintenance, metabolism, and autophagy [24][25][26][27][28][29][30]. ...
A small number of peptide growth factor ligands are used repeatedly in development and homeostasis to drive programs of cell differentiation and function. Cells and tissues must integrate inputs from these diverse signals correctly, while failure to do so leads to pathology, reduced fitness, or death. Previous work using the nematode C. elegans identified an interaction between the bone morphogenetic protein (BMP) and insulin/IGF-1-like signaling (IIS) pathways in the regulation of lipid homeostasis. The molecular components required for this interaction, however, were not fully understood. Here we report that INS-4, one of 40 insulin-like peptides (ILPs), is regulated by BMP signaling to modulate fat accumulation. Furthermore, we find that the IIS transcription factor DAF-16/FoxO, but not SKN-1/Nrf, acts downstream of BMP signaling in lipid homeostasis. Interestingly, BMP activity alters sensitivity of these two transcription factors to IIS-promoted cytoplasmic retention in opposite ways. Finally, we probe the extent of BMP and IIS interactions by testing additional IIS functions including dauer formation, aging, and autophagy induction. Coupled with our previous work and that of other groups, we conclude that BMP and IIS pathways have at least three modes of interaction: independent, epistatic, and antagonistic. The molecular interactions we identify provide new insight into mechanisms of signaling crosstalk and potential therapeutic targets for IIS-related pathologies such as diabetes and metabolic syndrome.
Aging encompasses the gradual and intrinsic progression of physical, psychological, and functional changes that occur in living organisms over time. In aged organisms, a noticeable reduction in histones levels is accompanied by both local and global chromatin remodeling. This shift creates an imbalance between activating and repressive histone modifications, resulting in widespread transcriptional changes observed in various aging models. Sirtuins, specifically class III NAD+-dependent histone deacetylases (HDAC), play a vital role in modifying various proteins, including histones, due to their deacetylase activity. These enzymes are intricately involved in critical epigenetic modifications and mediate a wide array of biological functions. Their roles encompass the maintenance of genomic stability, metabolism regulation, and apoptosis modulation, impacting the aging process, and even being implicated in tumorigenesis. Sirtuins possess the capability to recruit and activate several enzymes that are part of the epigenetic machinery. This includes DNA methyltransferases (DNMTs), histone acetyltransferases (HATs) and methyltransferases (HMTs). Consequently, sirtuins are pivotal in regulating heterochromatin formation and gene silencing. In addition to their epigenetic roles, sirtuins, particularly SIRT1, also interact with several key proteins, including NF-κB, AMPK, PGC1α, m-TOR, FOXOs and p53. These interactions are associated with various aspects of the aging process. SIRT1, a prominent member of the sirtuin family, has garnered considerable attention for its role in regulating longevity and its potential therapeutic applications in preventing and treating age-related diseases. In this chapter, we will delve into how epigenetic events influence the aging process and explore how SIRT1 can impact on longevity by inducing or modifying various epigenetic changes, including chromatin remodeling and histone acetylation and methylation.
Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.
IMPORTANCE
Human adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.
Extreme death feigning, longevity, and blue color make adult beetles of Asbolus verrucosus LeConte, 1851 (Coleoptera: Tenebrionidae) popular pets and demonstration insects. Additionally, autohemorrhaging in death-feigning larvae facilitates debris-based camouflage. The A. verrucosus genome was examined to gain insight into longevity and death feigning in this insect. A short-read assembly (∼250 Mbp) encoded > 90% of expected arthropod-specific proteins. The sex chromosomes represented 26.3% of the bivalent lengths and the haploid portion of the male genome (2n = 14; 6AA + Xy). Longevity-associated proteins for nutrient signaling and genome maintenance were present but require further investigation. For example, Sirtuin 6 harbored only two of the five amino acid changes known to enhance DNA repair and longevity. Comparative genomics identified non-synonymous substitutions in A. verrucosus proteins that were exclusive to long-duration death-feigning tenebrionids. The proteins are central to the development and function of neuromuscular junctions and may define a death-feigning pathway in insects. High-copy protein-coding contigs (3–300 copies/genome) were related to reflex bleeding (innate immunity and and melanization), longevity (response to stress, DNA repair, stem cell maintenance), and death feigning (neuronal development and signaling). Notably, telomerase-activating and stabilizing protein-coding regions were among the multicopy loci, along with loci encoding haponin and heat-shock proteins. It is tempting to conjecture that A. verrucosus is genetically poised for hormesis, where a small stress signal promotes an amplified protective response. The A. verrucosus genome is one of the few annotated tenebrionid genomes available and has impacted phylogenomic comparisons of insects and important gene families. The annotated genome was deposited into public databases under project PRJNA354432.
For the optimal survival of a species, an organism coordinates its reproductive decisions with the nutrient availability of its niche. Thus, nutrient-sensing pathways like insulin-IGF-1 signaling (IIS) play an important role in modulating cell division, oogenesis, and reproductive aging. Lowering of the IIS leads to the activation of the downstream FOXO transcription factor (TF) DAF-16 in Caenorhabditis elegans which promotes oocyte quality and delays reproductive aging. However, less is known about how the IIS axis responds to changes in cell cycle proteins, particularly in the somatic tissues. Here, we show a new aspect of the regulation of the germline by this nutrient-sensing axis. First, we show that the canonical G1-S cyclin, cyclin D/cyd-1, regulates reproductive aging from the uterine tissue of wild-type worms. Then, we show that knocking down cyd-1 in the uterine tissue of an IIS receptor mutant arrests oogenesis at the pachytene stage of meiosis-1 in a FOXO/DAF-16-dependent manner. We find that activated FOXO/DAF-16 destroys the somatic gonad tissues like the sheath cells, and transcriptionally prevents the spermatogenesis-to-oogenesis switch to orchestrate this arrest. Deleting FOXO/DAF-16 releases the arrest and restores the somatic gonad but leads to the production of poor-quality oocytes. Together, our study reveals the unrecognized cell non-autonomous interaction of CYD-1 and FOXO/DAF-16 in reproductive aging and the regulation of oogenesis.
Neurodegenerative diseases (NDs) are a heterogeneous group of aging-associated ailments characterized by interrupting cellular proteostasic machinery and the misfolding of distinct proteins to form toxic aggregates in neurons. Neurodegenerative diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and others, are becoming an increasing threat to human health worldwide. The degeneration and death of certain specific groups of neurons are the hallmarks of these diseases. Over the past decades, Caenorhabditis eleganshas beenwidely used as a transgenic model to investigate biological processes related to health and disease. The nematode Caenorhabditis elegans (C. elegans) has developed as a powerful tool for studying disease mechanisms due to its ease of genetic handling and instant cultivation while providing a whole-animal system amendable to several molecular and biochemical techniques. In this review, we elucidate the potential of C. elegans as a versatile platform for systematic dissection of the molecular basis of human disease, focusing on neurodegenerative disorders, and may help better our understanding of the disease mechanisms and search for new therapeutics for these devastating diseases.
The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-β-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.
Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of hpk-1 results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging hpk-1 expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system, hpk-1 induction overlaps with key longevity transcription factors, which suggests hpk-1 expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of hpk-1 extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requires mxl-2 (MLX), hlh-30 (TFEB), and daf-16 (FOXO). Our work establishes hpk-1 as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.
Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of hpk-1 results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging hpk-1 expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system, hpk-1 is co-expressed with key longevity transcription factors, including daf-16 (FOXO), hlh-30 (TFEB), skn-1 (Nrf2), and hif-1, which suggests hpk-1 expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of hpk-1 extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity. Our work establishes hpk-1 as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.
C. elegans insulin/insulin-like growth factor 1 signaling (IIS) affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its presence in all somatic cells, DAF-16's physiological effects, such as modulation of dauer formation, synapse maturation, axon regeneration, and adult longevity, exhibit prevalent tissue-specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to the functional diversity of IIS. To further examine this possibility, we sought to identify tissue-specific and direct transcriptional targets of DAF-16 in muscle cells. Following FACS-sorting to enrich mature muscle cells from young adult animals, we compared the muscle transcriptomes under high and low IIS signaling conditions, with and without DAF-16. We further analyzed and compared the DAF-16 docking sites in muscle and intestine cells from published datasets. These analyses revealed 14 potential muscle-specific DAF-16 transcriptional targets, among which we validated two that are strongly and specifically activated by DAF-16 in muscles: a secreted protein C54F6.5 and a calcium-binding protein CEX-1/Calexcitin. Both genes exhibit DAF-16-independent non-muscle expression, explaining their low rank or absence from the current DAF-16 target lists generated by multiple independent whole-animal microarray or mRNA-sequencing analyses. These results support the notion of tissue-specific DAF-16 transcriptional programs and highlight the importance of verifying FOXO targets in a cell-type-specific manner.
Riboflavin is an essential cofactor in many enzymatic processes and in the production of flavin adenine dinucleotide (FAD). Here, we report that the partial depletion of riboflavin through knockdown of the C. elegans riboflavin transporter 1 (rft‐1) promotes metabolic health by reducing intracellular flavin concentrations. Knockdown of rft‐1 significantly increases lifespan in a manner dependent upon AMP‐activated protein kinase (AMPK)/aak‐2, the mitochondrial unfolded protein response, and FOXO/daf‐16. Riboflavin depletion promotes altered energetic and redox states and increases adiposity, independent of lifespan genetic dependencies. Riboflavin‐depleted animals also exhibit the activation of caloric restriction reporters without any reduction in caloric intake. Our findings indicate that riboflavin depletion activates an integrated hormetic response that promotes lifespan and healthspan in C. elegans. Riboflavin depletion via knockdown of the C. elegans riboflavin transporter rft‐1 promotes longevity and metabolic hormesis. Riboflavin depletion mimics features of dietary restriction, and lifespan extension occurs through the activation of AMPK, FOXO, and the mitochondrial unfolded protein response.
Aging is the greatest risk factor for multiple leading causes of death, such as heart diseases, multiple types of cancer, and Alzheimer’s disease. Aging is thus a growing economic and health concern worldwide as the number of people over the age of 65 continues to increase. Multiple genetic and environmental pathways that slow aging have been discovered using animal models. However, the mechanisms by which these pathways extend lifespan remain largely unclear. Metabolism is a major regulator of longevity whose perturbation can slow aging and promote healthspan and longevity. Previous studies implicate metabolism in multiple longevity pathways, such as insulin signaling, nutrient sensing, and dietary restriction, across multiple organisms, including C. elegans. These findings make understanding the mechanisms of metabolic regulation of longevity a crucial next step. A major challenge to studying metabolism in C. elegans is its reliance on a live bacterial food source. Bacteria have their own metabolic activity that confounds the metabolic changes in C. elegans. While methods of killing bacteria to stop their metabolic activity exist, they make the bacteria inedible, present additional confounding variables or are not practical to use. Using paraformaldehyde (PFA), a crosslinking chemical, we developed a viable method of killing bacteria and stopping their metabolic activity that 1) is edible for the worms, 2) can be used in a high-throughput manner, and 3) does not substantially affect longevity phenotypes. Thus, PFA treatment is a viable way of preventing bacterial metabolism to study C. elegans metabolism. The PFA treatment allowed us to determine the metabolic changes that occur following the expression of fmo-2, a member of the highly conserved enzyme family flavin-containing monooxygenase and a major longevity regulator downstream of multiple pathways, including dietary restriction. Using metabolomics and RNAi knockdown, we determine that fmo-2 interacts with one carbon metabolism (OCM) to influence longevity and stress resistance. OCM is a metabolic network that has been implicated in multiple longevity pathways and is a crucial intermediate network for processes necessary for survival, including nucleotide synthesis, the transsulfuration pathway, and methylation. Using computational modeling, we identify the flux through methylation processes to be reduced in fmo-2 overexpression animals, suggesting that fmo-2 and reduced methylation flux are in the same functional pathway. Our data also identify tryptophan as an endogenous substrate of FMO-2 and implicate the kynurenine pathway as a target of FMO-2 that is likely linked to changes OCM. A potential downstream consequence of the changes in OCM following fmo-2 expression is the induction of endoplasmic reticulum unfolded protein response (UPRER), which is involved in proteostasis and longevity regulation. A reduction in methylation flux can lead to the reduction of phosphatidylcholine (PC) synthesis, which in turn can activate the UPRER. Our data thus far are consistent with a model where FMO-2 reduces PC synthesis to activate the UPRER, thereby promoting longevity and health. My thesis work furthers our understanding of a highly conserved enzyme family whose member serves as a critical convergence point for multiple longevity pathways. In addition, it also furthers our understanding of the metabolic regulation of the aging process and identifies key regulators that can potentially serve as therapeutic targets to slow aging to promote health and longevity.
Objective
To investigate how the environmental factor affects the delivery of nano silver through food chain, we set up a two-stage food delivery chain model of Escherichia coli and Caenorhabditis elegans system.
Methods
Through a two-stage food delivery chain model of E. coli and C. elegans, the mRNA expression levels of DAF-2, age-1, PDK-1, Akt-1 and DAF-16 in the insulin growth factor 1 signaling pathway in nematode gonad cells which occurs AgNPs induced apoptosis were evaluated and the apoptosis of gonad cells in the mutant strains of the above key genes were detected.
Results
DAF-2, age-1, PDK-1 and Akt-1 could significantly negatively regulate the apoptosis of nematode cells induced by AgNPs, while DAF-16 could significantly promote the apoptosis induced by AgNPs. The DAF-16 up-regulated expression was a protective effect on the body and the phenomenon of DNA double-strand breaks was significantly increased. The damage effect induced by AgNPs was significantly enhanced in the presence of the environmental factor fulvic acid.
Conclusion
The damage effect induced by AgNPs after food delivery involves the regulation of the insulin growth factor 1 signaling pathway and environmental factors have a significant impact on the biological effects.
Bay scallops are commercially important bivalve species for fisheries and aquaculture in China, the USA, Peru and Chile, but their small size, short lifespan (<14 months) and serious inbreeding depression restrict the sustainable development of their aquaculture. Compared with bay scallops, some interspecific hybrids of bay scallops and Peruvian scallops exhibit longer lifespans and significantly larger sizes, which may result from the longevity genes of Peruvian scallops, which have lifespans of 7–10 years. The FoxO transcription factor integrates signals from different longevity-related pathways that play a crucial role in aging and longevity in terrestrial model organisms. However, limited information is available on its roles in the longevity of marine animals. Here, we extended this paradigm using Peruvian scallops and bay scallops, which have distinct lifespans. Cloning and sequence analysis of the FoxO ORFs (ApFoxO and AiFoxO) revealed 22 synonymous, 7 nonsynonymous SNPs and two InDels between the two species. The amino acid variations (Q27 and E28) near the AKT phosphorylation site (T21) may affect the activities of ApFoxO for longevity. Nutrient restriction could extend lifespan in terrestrial model organisms, and the expression of ApFoxO and AiFoxO both significantly increased under nutrient restriction, but the response in bay scallops was faster and more robust than that in Peruvian scallops. Furthermore, ionizing radiation caused significantly lower mortality and higher FoxO expression in Peruvian scallops. These results suggested that FoxO may play a protective role in longevity through nutrient availability and DNA repair for genomic stability. The significantly decreased expression of CAT and Mn-SOD and the increased β-GAL activity (a marker for senescence) after FoxO downregulation suggested that FoxO may contribute to longevity by controlling ROS with its antioxidase activity. The results of our work provide the basis for understanding the role of FoxO in longevity in scallops and its potential as a biomarker for selecting larger hybrids to benefit scallop aquaculture.
p>Protein Kinase B (PKB), also known as Akt, was discovered in 1991 as the mammalian homologue of the viral oncogene product, v-akt. Subsequently, overexpression or constitutive activation of PKBα, PKBβ and PKBγ has been demonstrated in a number of cancers. The idea that inhibition of PKB in cancer cells may restore the sensitivity of cells to chemotherapeutic drugs has fuelled the search for specific inhibitors of PKB.
The aim was to manipulate PKB levels by overexpression or antisense technology to determine whether PKB regulates apoptosis. To test whether various PKB isoforms have the potential to regulate apoptosis in cells PKB levels were first modulated by overexpression. For this the activation of caspase-3 by staurosporine, an established induced or apoptosis, was employed. Overexpression of PKBα or PKBγ protected staurosporine-treated PC12 cells against the activation of caspase-3. To rigorously establish that these results were not non-specific effects of overexpression, antisense knockout of endogeneou PKB was then used to remove individual or combinations of PKB isoforms from cells. The combined depletion of greater than 95% PKBα, PKBβ and PKBγ from 3T3-L1 adipocytes sensitised cells to staurosporine-induced caspase-3 activation. Depletion of over 95% PKBα and PKBβ, or 95% of PKBγ alone, was not sufficient to sensitise 3T3-L1 adipocytes to the activation of caspase-3 induced by staurosporine. As staurosporine inhibited the activation of PKB, this effect requires PKB proteins and clearly indicates crucial roles of PKB in interacting with other proteins.
In conclusion these results show that endogenous PKB plays a critical role in regulating apoptosis as measured by caspase-3 activation. This antiapoptotic potential makes PKB an attractive drug target in cancer treatment.</p
The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.
Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMC) needed for vasodilation. While the regulation of NO-stimulated sGC activity has been well-characterized at the post-translational level, the mechanisms that govern sGC transcription remains incompletely understood. Recently, we identified forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC, however the specific FoxO family member responsible for expression of sGCβ in SMC remains unknown. Using FoxO shRNA knockdown adenoviruses, we show that FoxO1 or FoxO3 knockdown causes greater than 2-fold increases in GUCY1A3 and GUCY1B3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in GUCY1A3 and GUCY1B3 mRNA with 70% loss of sGCα and 50% loss of sGCβ protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter-luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of the GUCY1B3 gene. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCβ expression in SMC.
The chapter discusses the structure of protein kinase catalytic domains, and the classification of protein kinases according to the sequences of those domains. These enzymes use the gamma phosphate of ATP to generate phosphate monoesters utilizing protein alcohol groups, (on serine and threonine) and/or protein phenolic groups (on tyrosine) as phosphate group acceptors. They are related by virtue of their homologous “kinase domains” (also known as “catalytic domains”) which consist of 250–300 amino acid residues. In addition to mammals and other vertebrates, eukaryotic protein kinase superfamily members have been identified and characterized from a wide range of other animal phyla, plants, fungi, and microorganisms. Hence, the protein kinase progenitor gene can be traced back to a time prior to the evolutionary separation of the major eukaryotic kingdoms. In the future, it may be possible to model a three-dimensional structure of a novel protein kinase catalytic domain with sufficient accuracy to be able to predict the preferred primary sequence surrounding the hydroxyamino acid it phosphorylates, which in turn will allow one to predict what proteins might be its substrates from the increasingly complete database of protein sequences.
In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homologue of the mammalian insulin receptor) and AGE-1 (a homologue of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Here we show that null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-β-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human ortholognes of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-β effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes.
We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
The nematode Caenorhabditis elegans responds to conditions of overcrowding and limited food by arresting development as a dauer larva. Genetic analysis of mutations that alter dauer larva formation (daf mutations) is presented along with an updated genetic pathway for dauer vs. nondauer development. Mutations in the daf-2 and daf-23 genes double adult life span, whereas mutations in four other dauer-constitutive genes positioned in a separate branch of this pathway (daf-1, daf-4, daf-7 and daf-8) do not. The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determination of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan.
In situ hybridization on sections from the adult rat peripheral and central nervous systems demonstrated that trkB mRNA was expressed not only by neurons but also by cells in central nervous system white matter as well as by Schwann cells in the sciatic nerve. In situ hybridization with an oligonucleotide complementary to the trkB tyrosine kinase domain could only demonstrate mRNA in neurons, indicating expression of truncated trkB receptors lacking the tyrosine kinase domain by glial cells. RNA blot analysis was performed on separately cultured central nervous system glial cells to study which cell types express trkB mRNA. Several transcripts encoding truncated trkB receptors were expressed at high levels in O-2A progenitors, astrocytes, and oligodendrocytes, but not trkB mRNA could be detected in microglia. The expression of trkB mRNA by glial cells in vivo was also investigated after injury; strongly elevated levels of mRNA encoding truncated receptors were detected in the glial scar formed after an incision in the spinal cord dorsal funiculus. In contrast, in the cut sciatic nerve, trkB mRNA decreased distal to the transection, and by 3 weeks only very low levels of mRNA could be detected. Immunoelectron microscopy located trkB-like immunoreactivity to axons and Schwann cells in the sciatic nerve. The expression of truncated trkB receptors by astrocytes, oligodendrocytes, and Schwann cells and the altered levels in response to injury indicate that glial trkB receptors may serve an important function in the intact and injured nervous system.
Recessive mutations in two genes, daf-2 and age-1, extend the lifespan of Caenorhabditis elegans significantly. The daf-2 gene also regulates formation of an alternative developmental state called the dauer. Here we asked whether these two genes function in the same or different lifespan pathways. We found that the longevity of both age-1 and daf-2 mutants requires the activities of the same two genes, daf-16 and daf-18. In addition, the daf-2(e1370); age-1(hx546) double mutant did not live significantly longer than the daf-2 single mutant. We also found that, like daf-2 mutations, the age-1(hx546) mutation affects certain aspects of dauer formation. These findings suggest that age-1 and daf-2 mutations do act in the same lifespan pathway and extend lifespan by triggering similar if not identical processes.
The Caenorhabditis elegans dauer larva is specialized for dispersal without growth and is formed under conditions of overcrowding and limited food.
The daf-7 gene, required for transducing environmental cues that support continuous development with plentiful food, encodes a transforming
growth factor-β (TGF-β) superfamily member. A daf-7 reporter construct is expressed in the ASI chemosensory neurons. Dauer-inducing pheromone inhibits daf-7 expression and promotes dauer formation, whereas food reactivates daf-7 expression and promotes recovery from the dauer state. When the food/pheromone ratio is high, the level of daf-7 mRNA peaks during the L1 larval stage, when commitment to non-dauer development is made.
Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and
other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present studies, the constitutively active Akt and a nonmyristoylated
control mutant were expressed in 3T3-L1 cells that can be induced to differentiate into adipocytes. The constitutively active
Akt induced glucose uptake into adipocytes in the absence of insulin by stimulating translocation of the insulin-responsive
glucose transporter 4 to the plasma membrane. The constitutively active Akt also increased the synthesis of the ubiquitously
expressed glucose transporter 1. The increased glucose influx in the 3T3-L1 adipocytes directed lipid but not glycogen synthesis.
These results indicate that Akt can regulate glucose uptake and metabolism.
A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.
We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.
The influence of inositol phosphates and phosphoinositides on the α isoform of the RAC-protein kinase B (RAC/PKB) was studied
using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol
phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol
phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 μM and 0.5 μM, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the
fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol
3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate
was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 μM. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 μM). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate
that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products
of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide
further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.
Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase.
Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence
of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation
by the upstream kinase and it directly activated the upstream kinase.
A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the
long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases
in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor.
Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is
analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism,
diapause, and longevity.
In a 4-cell stage C. elegans embryo, signaling by the P2 blastomere induces anterior-posterior polarity in the adjacent EMS blastomere, leading to endoderm formation. We have taken genetic and reverse genetic approaches toward understanding the molecular basis for this induction. These studies have identified a set of genes with sequence similarity to genes that have been shown to be, or are implicated in, Wnt/Wingless signaling pathways in other systems. The C. elegans genes described here are related to wnt/wingless, porcupine, frizzled, beta-catenin/armadillo, and the human adenomatous polyposis coli gene, APC. We present evidence that there may be partially redundant inputs into endoderm specification and that a subset of these genes appear also to function in determining cytoskeletal polarity in certain early blastomeres.
The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced
activity of the genedaf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as
long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2mutations requires the activity of the gene daf-16. daf-16appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead
family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the
activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved.
The nematode Caenorhabditis elegans has two sexes, males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. We have now identified a binding factor (FBF) which is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. We propose that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins.
Recessive mutations in two genes, daf-2 and age-1, extend the lifespan of Caenorhabditis elegans significantly. The daf-2 gene also regulates formation of an alternative developmental state called the dauer. Here we asked whether these two genes function in the same or different lifespan pathways. We found that the longevity of both age-1 and daf-2 mutants requires the activities of the same two genes, daf-16 and daf-18. In addition, the daf-2(e1370); age-1(hx546) double mutant did not live significantly longer than the daf-2 single mutant. We also found that, like daf-2 mutations, the age-1(hx546) mutation affects certain aspects of dauer formation. These findings suggest that age-1 and daf-2 mutations do act in the same lifespan pathway and extend lifespan by triggering similar if not identical processes.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.
As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.
When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of organelle membranes, shape alteration through rearrangement of cytoskeletal actin, transformation and chemotaxis. But how PI(3)K mediates these responses is only now becoming clear.
The ‘pleckstrin homology’ domain is an approximately 100-residue protein module that has recently been added to the domain catalogue of signalling proteins. For this review we have made an extensive database search using a profile search method, and found a number of additional proteins that may contain PH domains. The PH domain is present in many kinases, isoforms of phospholipase C, GTPases, GTPase-activating proteins and nucleotide-exchange factors, including such proteins as Vav, Dbl and Bcr, and there are two PH domains in a guanine-nucleotide releasing factor of Ras. Many PH-domain-containing proteins interact with GTP-binding proteins. We have also identified a PH domain in β-adrenergic receptor kinase exactly in the region that has already been shown to be involved in binding to the β and γ subunits of a heterotrimeric G protein. This suggests that PH domains may be involved in interactions with GTP-binding proteins.
This chapter focuses on genetic mapping with polymorphic sequence-tagged sites (STSs), and its advantages. This mapping method offers a number of advantages over standard procedures. (1) It is rapid and requires only a single interstrain cross rather than a succession of crosses involving many different strains and requiring an elapsed time of several weeks. (2) STSs behave as dominant markers, permitting efficient and sensitive mapping strategies. In contrast, most conventional mapping in Caenorhabditis elegans uses recessive visible markers and less efficient strategies to detect linkage. (3) STSs can be scored in embryos. This permits efficient mapping of lethal mutations because the markers can be scored in lethal homozygotes that have arrested during embryonic development. (4) X-linked STS markers can be easily introduced into a mapping strain through males. In contrast, mapping on X with visible markers is often complicated by ineffective mating of hemizygous mutant males, and may require the construction of XX tra-l males. This mapping technique may permit new kinds of genetic analysis in C. elegans. One example is the mapping of multiple factors that contribute to quantitative traits.
ABSTRACT Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (α, β and γ) regulate transcription ofthe transthyretin (TTR)
and numerous other liver-specific genes. The HNF-3 proteins bind DNA via a homologous winged helixmotif common to a number
of develop mental regulatory proteins including the Drosophila homeotic fork head (fkh) protein. The mammalian HNF-3/fkh family consists of at least thirty distinct members and is expressed in a variety of different cellular lineages. In addition
to the winged helix motif, several HNF-3/fkh family members also share homology within transcriptional activation region II and III sequences. In the present study we
further define the sequence boundaries of the HNF-3β N-terminal transcriptional activation domain to extend from amino acids
14 to 93 and include conserved region IV and V sequences. We also demonstrate that activity of the HNF-3 N-terminal domain
was diminished by mutations which altered a putative α-helical structure located between amino acid residues 14 and 19. However,
transcriptional activity was not affected by mutations which eliminated two conserved casein kinase I sites or increased the
number of acidic amino acid residues in the N-terminal domain. Furthermore, we determined that the nuclear localization signal
overlaps with the winged helix DNA-binding motif. These results suggest that conserved sequences within the winged helix motif
of the HNF-3/fkh family maybe involved not only in DNA recognition, but also in nuclear targeting.
Glycogen synthase kinase-3 (GSK3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin, modulation of the transcription factors AP-1 and CREB, the specification of cell fate in Drosophila and dorsoventral patterning in Xenopus embryos. GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). Here we show, however, that agents which prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and we demonstrate that it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3 (refs 10, 14), the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase.
Larval development of the nematode Caenorhabditis elegans is controlled by the activities of four classes of chemosensory neurons. The choice between normal development and development into a specialized larval form called a dauer larva is regulated by competing environmental stimuli: food and a dauer pheromone. When the neuron classes ADF, ASG, ASI, and ASJ are killed, animals develop as dauer larvae regardless of environmental conditions. These neurons might sense food or dauer pheromone, or both, to initiate the specialized differentiation of many cell types that occurs during dauer formation. Entry into and exit from the dauer stage are primarily controlled by different chemosensory neurons. The analysis of mutants defective in dauer formation indicates that the chemosensory neurons are active in the absence of sensory inputs and that dauer pheromone inhibits the ability of these neurons to generate a signal necessary for normal development.
The free-living nematode Caenorhabditis elegans is used as a genetically manipulable experimental system for the study of aging. Utilizing a temperature-sensitive sterile strain with a normal life span, a method is described for the isolation of mutant strains with significantly increased life spans. Eight mutant strains were isolated each having increased life spans. Two mutant strains were spontaneous dauer formers, accounting for their increased longevity. Another was chemotaxis-defective, causing reduced food intake which could account for its increased life span. Five mutants suffered from varying degrees of paralysis affecting their rate of pharyngeal pumping and food ingestion. The high correlation of the decreased rate of food ingestion of these mutants with their increased longevity is interpreted as indicating that the increased longevity is most likely due to reduced caloric intake. These results appear to indicate that specific life span genes are extremely rare or, alternatively, life span is controlled in a polygenic fashion.
The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.
In summary, type II diabetes is a disease with a very slow and progressive pathogenesis. Both genes and the environment play a critical role in development of this disease. In genetically prone individuals, insulin resistance is the earliest detectable defect. This defect may occur 15-25 years or more before the clinical onset of disease. Thus, insulin resistance constitutes what epidemiologists call an 'intervening phenotype,' as well as a marker for the disease. The exact molecular site of the insulin resistance is unknown, but even early in the pathogenesis there are multiple alterations in the insulin action cascade. Initially, there is an attempt to compensate for this resistance with increased insulin secretion, but eventually, insulin secretion fails and type II diabetes develops. This cascade of events is programmed by a series of diabetes-causing genes, or diabetogenes. Some of these diabetogenes may be primary and actually cause the insulin resistance, whereas others may be related to the diabetic state and be secondary. Whether our new diabetogene, Rad, is primary or secondary remains to be determined, but in either case, Rad may be a useful marker of the disease. Environmental factors, particularly those leading to obesity, further enhance this diabetogenic tendency by accentuating the insulin resistance. Thus, as illustrated in the cartoon by my friend Pierre DeMeyts shown in Fig. 23, our search for the type II diabetes gene, at least up to now, has been much like the blind man trying to describe an elephant. Each of us has our own candidate genes of interest, each of us has our own perspective of what might be the most important lesion. However, through the increased use of cell biological and molecular biological techniques coupled with physiological data and clinical insights, we are now closer than ever to solving this problem; closer than ever to piecing together, not only the story of insulin action, but the probable cause of defects in insulin action in type II diabetes. The search for the cause of type II diabetes is not an easy one. But as pointed out by Sir Frederick Banting, no matter how difficult a problem, 'you must begin with an ideal and end with an ideal.' Finally, I wish to close this Banting Lecture by giving my thanks to my mentors, fellows, colleagues and coworkers (listed in the ACKNOWLEDGMENTS) without whose help this work would not have been possible, as well as to the members and leadership of the American Diabetes Association for their support of diabetes research and for giving me this award. As we hear on TV, there is a greeting card for every occasion, and this one is no exception, so here is my card to you: I thank you from the bottom of my pancreas. That's like the bottom of my heart, only deeper.
Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.
Currently, a central question in biology is how signals from the cell surface modulate intracellular processes. In recent years phosphoinositides have been shown to play a key role in signal transduction. Two phosphoinositide pathways have been characterized, to date. In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphospate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators. The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake. Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking.
In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined.
The 'pleckstrin homology' domain is an approximately 100-residue protein module that has recently been added to the domain catalogue of signalling proteins. For this review we have made an extensive database search using a profile search method, and found a number of additional proteins that may contain PH domains. The PH domain is present in many kinases, isoforms of phospholipase C, GTPases, GTPase-activating proteins and nucleotide-exchange factors, including such proteins as Vav, Dbl and Bcr, and there are two PH domains in a guanine-nucleotide releasing factor of Ras. Many PH-domain-containing proteins interact with GTP-binding proteins. We have also identified a PH domain in beta-adrenergic receptor kinase exactly in the region that has already been shown to be involved in binding to the beta and gamma subunits of a heterotrimeric G protein. This suggests that PH domains may be involved in interactions with GTP-binding proteins.
We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. Both genes also regulate formation of the dauer larva, a developmentally arrested larval form that is induced by crowding and starvation and is very long-lived. Our findings raise the possibility that the longevity of the dauer is not simply a consequence of its arrested growth, but instead results from a regulated lifespan extension mechanism that can be uncoupled from other aspects of dauer formation. daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.
A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13 degrees bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three alpha-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel beta-structure and random coil that interacts with the minor groove.
A pheromone-induced neurosecretory pathway in Caenorhabditis elegans triggers developmental arrest and an increase in longevity at the dauer diapause stage. The gene age-1 is required for non-dauer development and normal senescence. age-1 encodes a homologue of mammalian phosphatidylinositol-3-OH kinase (PI(3)K) catalytic subunits. Lack of both maternal and zygotic age-1 activity causes dauer formation, whereas animals with maternal but not zygotic age-1 activity develop as non-dauers that live more than twice as long as normal. These data suggest that phosphatidylinositol signalling mediated by AGE-1 protein controls lifespan and the dauer diapause decision.
Recently, a number of cDNA clones with homology to the catalytic subunit of phosphoinositide 3-kinase have been identified, and the sequence of the first cDNA clone encoding a phosphatidylinositol 4-phosphate 5-kinase has been published. Use of both dominant-negative mutants of phosphoinositide 3-kinase and the inhibitors wortmannin and LY294002 has identified a number of processes in which phosphoinositide 3-kinase participates, including cell motility, the Ras pathway, vesicle trafficking and secretion, and apoptosis. Several possible biochemical targets of phosphoinositides have been found.
Formation of the C. elegans dauer larva is repressed by the chemosensory neurons ADF, ASI, and ASG. Mutant analysis has defined two parallel genetic pathways that control dauer formation. By killing neurons in these mutants, we show that mutations in one of these genetic pathways disrupt dauer repression by ADF, ASI, and ASG. One gene in this pathway is daf-7, which encodes a TGFbeta-related protein. We find that daf-7::GFP fusions are expressed specifically in ASI and that expression is regulated by dauer-inducing sensory stimuli. We also show that a different chemosensory neuron, ASJ, functions in parallel to these neurons to induce dauer formation. Mutations in the second genetic pathway activate dauer formation in an ASJ-dependent manner. Thus, the genetic redundancy in this process is reflected at the neuronal level.
Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.
Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).
The substrate specificity of protein kinase-B alpha (PKBalpha, also known as RAC kinase or Akt) was investigated using synthetic peptide substrates related to the sequence surrounding the phosphorylation site on glycogen synthase kinase-3 (GSK3). The minimum sequence motif required for efficient phosphorylation was Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyd, where Xaa is any amino acid, Yaa and Zaa are small residues other than glycine and Hyd is a bulky hydrophobic residue (Phe, Leu). The most effective substrate, Arg-Pro-Arg-Thr-Ser-Ser-Phe, was phosphorylated with a Km of 5 microM and Vmax of 260 U/mg. PKBalpha phosphorylated histone H2B (Km 5 microM, Vmax 68 U/mg) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. The peptide Arg-Pro-Arg-Ala-Ala-Thr-Phe may be a relatively specific substrate for PKBalpha because, unlike other substrates, it is not phosphorylated by p70 S6 kinase or MAP kinase activated protein (MAPKAP) kinase-1.
The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated.
Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain
was not activated by PI 3-kinase in vivo or by PtdIns-3,4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity
than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.
The viability of vertebrate cells depends on survival factors which activate signal transduction pathways that suppress apoptosis. Defects in anti-apoptotic signalling pathways are implicated in many pathologies including cancer, in which apoptosis induced by deregulated oncogenes must be forestalled for a tumour to become established. Phosphatidylinositol-3-kinase (PI(3)K) is involved in the intracellular signal transduction of many receptors and has been implicated in the transduction of survival signals in neuronal cells. We therefore examined the role of PI(3)K, its upstream effector Ras, and its putative downstream protein kinase effectors PKB/Akt and p70S6K (ref. 5) in the modulation of apoptosis induced in fibroblasts by the oncoprotein c-Myc. Here we show that Ras activation of PI(3)K suppresses c-Myc-induced apoptosis through the activation of PKB/Akt but not p70S6K. However, we also found that Ras is an effective promoter of apoptosis, through the Raf pathway. Thus Ras activates contradictory intracellular pathways that modulate cell viability. Induction of apoptosis by Ras may be an important factor in limiting the expansion of somatic cells that sustain oncogenic ras mutations.
BACKGROUND: Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown. RESULTS: We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. We tested the kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) or PtdIns(3,4)P2 were effective in potently activating the kinase, which has been named PtdIns(3,4,5)P3-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. CONLCUSIONS: PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4, 5)P3 and/or PtdIns(3,4)P2.
When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of organelle membranes, shape alteration through rearrangement of cytoskeletal actin, transformation and chemotaxis. But how PI(3)K mediates these responses is only now becoming clear.
Signals from TGF-beta superfamily receptors are transduced to the nucleus by Smad proteins, which transcriptionally activate target genes. In Caenorhabditis elegans, defects in a TGF-beta-related pathway cause a reversible developmental arrest and metabolic shift at the dauer larval stage. Null mutations in daf-3 suppress mutations in genes encoding this TGF-beta signal, its receptors, and associated Smad signal transduction proteins. daf-3 encodes a Smad protein that is most closely related to mammalian DPC4, and is expressed throughout development in many of the tissues that are remodeled during dauer development. DAF-4, the type II TGF-beta receptor in this pathway, is also expressed in remodeled tissues. These data suggest that the DAF-7 signal from sensory neurons acts as a neuroendocrine signal throughout the body to directly regulate developmental and metabolic shifts in tissues that are remodeled during dauer formation. A full-length functional DAF-3/GFP fusion protein is predominantly cytoplasmic, and this localization is independent of activity of the upstream TGF-beta-related pathway. However, this fusion protein is associated with chromosomes in mitotic cells, suggesting that DAF-3 binds DNA directly or indirectly. DAF-3 transgenes also interfere with dauer formation, perhaps attributable to a dosage effect. A truncated DAF-3/GFP fusion protein that is predominantly nuclear interferes with dauer formation, implying a role for DAF-3 in the nucleus. These data suggest that DAF-7 signal transduction antagonizes or modifies DAF-3 Smad activity in the nucleus to induce reproductive development; when DAF-7 signals are disabled, unmodified DAF-3 Smad activity mediates dauer arrest and its associated metabolic shift. Therefore, daf-3 is unique in that it is antagonized, rather than activated, by a TGF-beta pathway.