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Retracted: Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity
Abstract
The development of a polarised morphology with multiple dendrites and a single axon is an essential step in the differentiation of neurons. The establishment of neuronal polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process during the establishment of neuronal polarity where it specifies axonal identity. Here, we show that the ubiquitin ligases Smad ubiquitination regulatory factor-1 (Smurf1) and Smurf2 are essential for neurite growth and neuronal polarity, respectively, and regulate the GTPases Rho and Rap1B in hippocampal neurons. Smurf2 is required for the restriction of Rap1B to a single neurite. Smurf2 ubiquitinates inactive Rap1B and initiates its degradation through the ubiquitin/proteasome pathway (UPS). Degradation of Rap1B restricts it to a single neurite and thereby ensures that neurons extend a single axon.
... Protein degradation is also involved in C3G functions, since the ubiquitination of Crk perturbs its binding to C3G, leading to decreased Rap1 activity (Shao et al., 2003 ). Likewise , it was reported that activation of Rap1 protects it from ubiquitination and proteasome degradation (Schwamborn et al., 2007). Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/ threonine kinase belongs to the family of Cdks. ...
... We found that only roscovitine affects the overall levels of Rap1 protein (Fig. 1C ). Because of Rap1 expression can be regulated by proteasome degradation (Schwamborn et al., 2007), we evaluated the effect of a proteasome inhibitor, MG-132, over Rap1 expression. COS-7 cells co-transfected with C3G, pBI-p35/EGFP, and tTA were treated with roscovitine with or whithout MG-132 (20 lM) during 18 h. ...
... Therefore, it is plausible to propose that the Reelin signaling pathway may induce C3G serine phosphorylation by Cdk5. It is known that Rap1 protein is regulated by Smurf2 ubiquitination and subsequent degradation by a proteasome pathway (Schwamborn et al., 2007). It is worth noting that Rap1 protein levels were also regulated by the Cdk5 activity, suggesting that Cdk5-mediated C3G phosphorylation stabilizes Rap1 protein against proteosome degradation.. Interestingly, the effect of MG- 132, a proteasome inhibitor, produced an increased in Rap1 protein level in COS-7 cells treated with roscovitine, suggesting that Cdk5 may regulated Rap1 degradation. ...
... The Smurf1-related E3 ubiquitin ligase Smurf2 also regulates the polarization of hippocampal neurons [47]. Smurf2 is expressed in neuronal processes as well as the soma in hippocampal neurons. ...
... Smurf2 induces the ubiquitination and consequent degradation of the GTPase Rap1B. Rap1B is enriched within the growth cone of the neuronal process fated to become the axon, and Rap1B stimulates axon specification in hippocampal neurons [47]. Deletion of Smurf2 in hippocampal neurons leads to the ectopic expression of Rap1B in multiple neuronal processes, thereby inducing supernumerary axons [47]. ...
... Rap1B is enriched within the growth cone of the neuronal process fated to become the axon, and Rap1B stimulates axon specification in hippocampal neurons [47]. Deletion of Smurf2 in hippocampal neurons leads to the ectopic expression of Rap1B in multiple neuronal processes, thereby inducing supernumerary axons [47]. Thus, Smurf1 and Smurf2 may act in opposing fashions locally in neuronal processes undergoing axon specification. ...
Recent studies have revealed that E3 ubiquitin ligases have essential functions in the establishment of neuronal circuits. Strikingly, a common emerging theme in these studies is that spatial organization of E3 ubiquitin ligases plays a critical role in the control of neuronal morphology and connectivity. E3 ubiquitin ligases localize to the nucleus, centrosome, Golgi apparatus, axon and dendrite cytoskeleton, and synapses in neurons. Localization of ubiquitin ligases within distinct subcellular compartments may facilitate neuronal responses to extrinsic cues and the ubiquitination of local substrates. Here, we review the functions of neuronal E3 ubiquitin ligases at distinct subcellular locales and explore how they regulate neuronal morphology and function in the nervous system.
... The formation of the neuronal networks present in the adult nervous system is a highly dynamic and tightly regulated process, involving the development of neurons with polarised morphology, axon pathfinding, and the establishment of an intricate network of synaptic connections. The UPS has emerged as a crucial component in the regulation of the developing nervous system [49] by controlling the proliferation of neuronal precursors and cell specification5051525354, neuronal migration [55, 56], neuritogenesis57585960616263, and synaptic pruning646566. E3 ligases are particularly relevant during the development of the nervous system, selecting key regulators for posterior proteasomal degradation or directing these proteins for specific signaling cascades. ...
... Given the key role of the UPS in the regulation of the protein content in the cytoplasm and in the nucleus, it is not surprising that it also contributes to this and other aspects of neuronal development. Thus, during the establishment of neuronal polarity in cultured rat hippocampal neurons, the degradation of Rap1b-GTPase by the UPS, mediated by E3 Smad ubiquitylation regulatory factors 1 and 2 (Smurf1 and Smurf2), defines which neurites become the axon and which are dismantled; only neurites with an accumulation of active Rap1B-GTP are viable [60]. The axon guidance cues netrin-1 and semaphorin 3A are involved in the regulation of growth cone guidance both in Xenopus [70, 71] as well as in mammals [72]. ...
... The interaction between these proteins is thought to activate the GLO pathway, with RPM-1 acting as a positive regulator, promoting vesicular trafficking essential for correct axon termination and synaptogenesis in mechanosensory neu- rons [79]. RPM-1 forms a SCF-like complex E3 ligase with the F-box protein FSN-1, the SKP1 ortholog SKR-1, and the Cullin CUL-1 [80], and besides its role in the regulation [50] HUWE1 HUWE1 supression of a N-Myc-DLL3 cascade, setting cell-cycle withdrawal and neuronal differentiation, restrains proliferation and enables neuronal differentiation [53] BTBD6 BTBD6 acts as an adaptor protein in the SCF E3 ligase complex and targets the transcriptional repressor and neurogenesis inhibitor Plzf for degradation [54] MIB1 RING-type E3 MIB1 expressing cells generate Notch signaling in neighboring radial glial cells to maintain their stemness and correct differentiation [52] TRIM11 TRIM11 mediates the degradation of the development regulator transcription factor Pax6 [51] Neural cell migration E3 ligase complex containing cullin 5 Cul5 auxiliates Dab1 dgradation in target neurons after a signaling cascade that involves VLDR and ApoE receptors, directing the speed and correct migration of neuronal populations [55, 56] Axonal growth RPM-1 RPM-1 E3 ligase negatively regulates axon outgrowth by the guidance receptors SAX-3/robo and UNC-5/UNC-5 [78] CDH1-APC complex Ubiquitin ligase CDH1-APC operates in the nucleus of neurons to inhibit axon growth through the promotion of transcription factors SnoN and Id2 degradation [58, 59] NEDD4 NEDD4 acts as a positive regulator of dendrite extension and arborization through the ubiquitination of RAP2A and PTEN downregulation [61, 62] SMURF1 SMURF1 enhances neurite outgrowth ubiquitinating RhoA [57] SMURF2 SMURF2 ubiquitinates RAP1B directing it to proteasomal degradation, assuring that only one neurite will become an axon [60] Synaptic pruning DIAP1 E3 ligase DIAP1 degradation, mediated by UBCD1 conjugating enzyme, leads to a caspase-dependent efficient pruning of the C4da neuron in Drosophila [65] RPM-1 RPM-1 E3 ligase negatively regulates a p38 MAPK pathway contibuting to the correct formation of mature synapses in C. elegans [64] SKR1 SKR-1, a core component of SCF E3, contributes to synapse elimination in a proteasome-dependent manner in HSNL [66] UPS in the nervous system of axon outgrowth, RPM-1 also acts as a negative modulator of a MAP kinase cascade that regulates pre-synaptic architecture, including the Dual-Leucine zipper Kinase MAPKKK (DLK-1), MKK-4, and the p38 MAPK ortholog, PMK-3 [64]. In the context of nervous system development, DUBs also play an important role, with relevant examples in most DUB classes. ...
In addition to its central roles in protein quality control, regulation of cell cycle, intracellular signaling, DNA damage response and transcription regulation, the ubiquitin-proteasome system (UPS) plays specific roles in the nervous system, where it contributes to precise connectivity through development, and later assures functionality by regulating a wide spectrum of neuron-specific cellular processes. Aberrations in this system have been implicated in the etiology of neurodevelopmental and neurodegenerative diseases. In this review, we provide an updated view on the UPS and highlight recent findings concerning its role in normal and diseased nervous systems. We discuss the advantages of the model organism Caenorhabditis elegans as a tool to unravel the major unsolved questions concerning this biochemical pathway and its involvement in nervous system function and dysfunction, and expose the new possibilities, using state-of-the-art techniques, to assess UPS function using this model system.
... Expression of the E3 ubiquitin ligase, Smurf2, had the most significant effect on EZH2 protein levels compared with the other E3 ubiquitin ligases (Mdm2, Fbw7, Cbl-b, Skp2 and Smurf1), and Smurf2-mediated decrease in EZH2 was restored by the addition of a proteasome inhibitor, MG132 (Supporting InformationFig S2), suggesting that Smurf2 is the primary E3 ligase for EZH2. Smurf2 is required for the establishment of neuronal polarity (Schwamborn et al, 2007). We found that its mRNA (Fig 2B) and protein levels (Fig 2C) were increased during neuron differentiation . ...
... Smurf2 is related to ubiquitin E3 ligases of the C2-WW-HECT family that play an important role in signalling regulation and planar cell polarity and motility (Izzi & Attisano, 2006; Narimatsu et al, 2009). It has been suggested that Smurf2 targets Smad1, Smad2, Smad7 and the TGF-b receptor for degradation and is involved in the establishment of neuronal polarity via ubiquitination of the GTPase Rap1B (Kavsak et al, 2000; Lin et al, 2000; Schwamborn et al, 2007; Zhang et al, 2001). In particular, mice with the Smurf2 mutant display defects that include failure to close the neural tube (Narimatsu et al, 2009). ...
EZH2 plays an important role in stem cell renewal and maintenance by inducing gene silencing via its histone methyltransferase activity. Previously, we showed that EZH2 downregulation enhances neuron differentiation of human mesenchymal stem cells (hMSCs); however, the underlying mechanisms of EZH2-regulated neuron differentiation are still unclear. Here, we identify Smurf2 as the E3 ubiquitin ligase responsible for the polyubiquitination and proteasome-mediated degradation of EZH2, which is required for neuron differentiation. A ChIP-on-chip screen combined with gene microarray analysis revealed that PPARγ was the only gene involved in neuron differentiation with significant changes in both its modification and expression status during differentiation. Moreover, knocking down PPARγ prevented cells from undergoing efficient neuron differentiation. In animal model, rats implanted with intracerebral EZH2-knocked-down hMSCs or hMSCs plus treatment with PPARγ agonist (rosiglitazone) showed better improvement than those without EZH2 knockdown or rosiglitazone treatment after a stroke. Together, our results support Smurf2 as a regulator of EZH2 turnover to facilitate PPARγ expression, which is specifically required for neuron differentiation, providing a molecular mechanism for clinical applications in the neurodegenerative diseases.
... Overexpression of constitutively active Rap1B rescues the loss of polarity after treatment with a PI3K inhibitor, indicating that Rap1B is one of the main targets of this pathway. The restriction of Rap1B to a single neurite is mediated by the UPS (Schwamborn et al. 2007a). Inactive, GDP-bound Rap1B is ubiquitinated by the ubiquitin E3 ligase Smurf2, which initiates its destruction through the proteasome. ...
Neurons are highly polarized cells that form distinct axonal and somatodendritic compartments. The establishment of this neuronal polarity, i.e., the specification of an axon and multiple dendrites, is essential for the normal structure and function of the nervous system. During embryonic development, proliferation, asymmetric division, and migration transform a single layer of highly polarized neuronal precursors into a structure with six distinct layers that is characteristic for the mammalian neocortex. The neuronal progenitor cells in the ventricular zone of the brain serve as the major source of pyramidal neurons in the telencephalon. Postmitotic neurons become polarized during their migration from the ventricular zone to the cortical plate by extending a leading process and a trailing axon. However, neuronal development is difficult to analyze in situ and requires advanced microscopy setups for imaging. Therefore, cultures of dissociated neurons have been instrumental in identifying the pathways that direct the establishment of neuronal polarity. These cultures allow to observe neuronal differentiation in an accessible and homogeneous environment with reduced complexity. In this chapter, we will discuss factors required for the establishment of neuronal polarity that were identified using cultured neurons and the extent to which their physiological function has been confirmed by the analysis of knockout mice.
... Using in vivo ubiquitination studies, we demonstrated that in H441 cells, overexpression of wild-type SMURF2 enhances β-TrCP1 polyubiquitination, whereas overexpression of catalytically inactive SMURF2 (CA) mutant inhibits it (Figure 3F ). Furthermore, as SMURF2 is known to partner with different E2s including UBCH7 and UBCH5 [35,37], we examined the involvement of SMURF2 ubiquitination machinery components in β-TrCP1 polyubiquitination using in vitro ubiquitination assay. Results obtained from this study demonstrate enhanced polyubiquitination of β-TrCP1 in the presence of purified SMURF2 and UBCH5 (Figure 3G). ...
Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.
... Smurf2 interacts with phosphorylated Smads and induces their rapid ubiquitination and degradation (Lo and Massague, 1999; Gao et al., 2009; David et al., 2013 ). Subsequent studies extended the repertoire of Smurf2 substrates and show a broader spectrum of Smurf2 biological functions (Li and Seth, 2004; Schwamborn et al., 2007). Induction in Smurf2 expression is linked to telomere attrition, and forced expression of Smurf2 enhances senescence in human fibroblasts (Zhang et al., 2004 ). ...
Smad ubiquitin regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that regulates TGF-β/Smad signaling and is implicated in a wide variety of cellular responses, but the exact mechanisms that control Smurf2 abundance remain largely unknown. Here we identified microRNA-322 (miR-322) and miR-503 as novel factors that regulate Smurf2 expression posttranscriptionally. Both miR-322 and miR-503 interacted with Smurf2 mRNA via its 3'-untranslated region (UTR) and repressed Smurf2 translation but did not affect total Smurf2 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-322/503 through a single binding site in the Smurf2 3'-UTR, whereas point mutation of this site prevented miR-322/503-induced repression of Smurf2 translation. Increased levels of endogenous Smurf2 by antagonization of miR-322/503 inhibited TGF-β-induced Smad2 activation by increasing the degradation of phosphorylated Smad2. Furthermore, the increase in Smurf2 in intestinal epithelial cells (IECs) expressing lower levels of miR-322/503 was associated with increased resistance to apoptosis, which was abolished by Smurf2 silencing. These findings indicate thatmiR-322/503 represses Smurf2 translation, in turn affecting intestinal epithelial homeostasis by altering TGF-β/Smad2 signaling and IEC apoptosis.
... Second, axon selective accumulation of these proteins can be the consequence of protein degradation in other neurites. In this sense, E3-ubiquitin ligase Smurf2 initiates the degradation of the Rap1b GTPase in all neurites of unpolarized neuron stage 2, except in the future axon [10] controlling proper neuronal polarization . Moreover, the E3-ubiquitin ligase Smurf1 when is phosphorylated by PKA changes substrate affinity contributing to RhoA degradation and accumulation of the Par6 protein at the axon tip, contributing to axon initiation and proper polarity formation [11]. ...
Chaperones are critical for the folding and regulation of a wide array of cellular proteins. Heat Shock Proteins (Hsps) are the most representative group of chaperones. Hsp90 represents up to 1-2% of soluble protein. Although the Hsp90 role is being studied in neurodegenerative diseases, its role in neuronal differentiation remains mostly unknown. Since neuronal polarity mechanisms depend on local stability and degradation, we asked whether Hsp90 could be a regulator of axonal polarity and growth. Thus, we studied the role of Hsp90 activity in a well established model of cultured hippocampal neurons using an Hsp90 specific inhibitor, 17-AAG. Our present data shows that Hsp90 inhibition at different developmental stages disturbs neuronal polarity formation or axonal elongation. Hsp90 inhibition during the first 3hours in culture promotes multiple axons morphology, while this inhibition after 3hours slows down axonal elongation. Hsp90 inhibition was accompanied by decreased Akt and GSK3 expression, as well as, a reduced Akt activity. In parallel, we detected an alteration of kinesin-1 subcellular distribution. Moreover, these effects were seconded by changes in Hsp70/Hsc70 subcellular localization that seem to compensate the lack of Hsp90 activity. In conclusion, our data strongly suggests that Hsp90 activity is necessary to control the expression, activity or location of specific kinases and motor proteins during the axon specification and axon elongation processes. Even more, our data demonstrate the existence of a "time-window" for axon specification in this model of cultured neurons after which the inhibition of Hsp90 only affects axonal elongation mechanisms.
... Smurf1 and Smurf2 share 73% identity at the amino acid level, and it is becoming clear that these ubiquitin ligases have both common and distinct functions in Xenopus development, while there appears to be more redundancy in mice. Smurfs have been shown to ubiquitinate and regulate several molecules involved in cell adhesion and migration (e.g., RhoA, Rap1, talin, hPEM-2, and Pk1) (Schwamborn et al. 2007; Narimatsu et al. 2009; Huang 2010). Smurf1 is localized to lamellipodia and filopodia and can induce protrusions (Wang et al. 2003; Huang et al. 2009), while overexpression or loss of function of Smurf2 promotes or inhibits the migration of some cell lines (Jin et al. 2009; Yang et al. 2009). ...
The formation of tissue boundaries is dependent on the cell-cell adhesion/repulsion system that is required for normal morphogenetic processes during development. The Smad ubiquitin regulatory factors (Smurfs) are E3 ubiquitin ligases with established roles in cell growth and differentiation, but whose roles in regulating cell adhesion and migration are just beginning to emerge. Here, we demonstrate that the Smurfs regulate tissue separation at mesoderm/ectoderm boundaries through antagonistic interactions with ephrinB1, an Eph receptor ligand that has a key role in regulating the separation of embryonic germ layers. EphrinB1 is targeted by Smurf2 for degradation; however, a Smurf1 interaction with ephrinB1 prevents the association with Smurf2 and precludes ephrinB1 from ubiquitination and degradation, since it is a substantially weaker substrate for Smurf1. Inhibition of Smurf1 expression in embryonic mesoderm results in loss of ephrinB1-mediated separation of this tissue from the ectoderm, which can be rescued by the coincident inhibition of Smurf2 expression. This system of differential interactions between Smurfs and ephrinB1 regulates the maintenance of tissue boundaries through the control of ephrinB protein levels.
... Furthermore, we had observed a decrease in the total Rho2 level of cells lacking Rga2, Rga7 and Rga6, suggesting that GTP-Rho2 is also regulated by degradation. Some examples of Rho regulation by protein degradation have been described in animal cells (Wang et al., 2003; Schwamborn et al., 2007 ), and it has been recently published that Rdi, the only S. cerevisiae Rho GDI, regulates Rho4 degradation by a proteolytic pathway that includes the proteasome, vacuolar proteases and the GSK-3b homologue Ygk3 (Tiedje et al., 2008 ). Additional experiments would be required to study the hypothethical Rho2 regulation by degradation. ...
Schizosaccharomyces pombe Rho2 GTPase regulates alpha-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2Delta cells contain more alpha-D-glucan and therefore partially suppress the thermosensitivity of mok1-664 cells, which have a defective alpha-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2-Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway.
... The Rab subfamily of small GTPases consists of 57 members in A. thaliana, most of which are involved in vesicle trafficking (Vernoud et al. 2003). The interaction between APD2 and the Rab homologue E1b implies a possible link between E3 ligases and small GTPases in plants, which was previously reported only in animals (Wilkins et al. 2004; Moren et al. 2005; Jura et al. 2006; Schwamborn et al. 2007; Berthold et al. 2008; Kim et al. 2009; Kawabe et al. 2010). In conclusion, we provide evidence to show that the four APDs, APD1, 2, 3 and 4, play important but redundant roles in the PMII process during male gametogenesis. ...
Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the reproductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant Pollen Development 1 (APD1) to APD4, as regulators of pollen mitosis II (PMII) in Arabidopsis thaliana (L.). The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis.
... Moreover , Smurfs have additional substrates that are not involved in the TGF-b pathway. By regulating stability and/or activities of their substrate proteins, Smurfs participate in a variety of biological processes, such as cell proliferation, senescence, migration, differentiation, and polarity (Zhao et al., 2003Zhao et al., , 2010 Yamashita et al., 2005; Sahai et al., 2007; Schwamborn et al., 2007; Cheng et al., 2011; Kong et al., 2011). The roles of Smurfs during ...
Smad ubiquitination regulatory factor (Smurf) 1 and 2 are E3 ubiquitin ligases originally identified as inhibitors of transforming growth factor beta signaling and are shown to modulate multiple cellular activities. The roles of Smurfs in vertebrate embryogenesis, however, are not completely understood.
Here we investigate the function of Smurf2 during early Xenopus development. We show that distinctly from Smurf1, overexpression of Smurf2 in presumptive mesoderm interfered with mesoderm induction and caused axial defects, whereas knockdown of Smurf2 with antisense morpholino oligonucleotides resulted in expansion of the mesoderm. These results imply that Smurf2 may modulate nodal-mediated mesodermal induction. Consistently, ventral expression of Smurf2 induced a partial secondary axis with head structures. In the ectoderm, Smurf2 resembled Smurf1 in controlling neural and epidermal marker expression and influencing head formation. Smurf1, but not Smurf2, additionally affected neural tube closure. Interestingly, both Smurfs could enhance as well as repress neural crest markers, implying that they modulate their targets dynamically during neural plate border specification.
Our data demonstrate that Smurf1 and Smurf2 have overlapping and distinct functionalities during early frog embryogenesis; collectively, they regulate ectodermal and mesodermal induction and patterning to ensure normal development of Xenopus embryos.
Molecular chaperones often work collaboratively with the ubiquitination-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development, and neuronal functions, and these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and cochaperones. MEC-15 likely functions in a SCF complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and chaperones; this antagonism regulates TRN development as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between UPS and chaperones tightly controls neuronal differentiation.
Heat shock proteins (Hsp) are primarily protecting and maintaining cell viability during stressful conditions such as thermal and oxidative challenges through protein refolding and stabilization. Hsp play an essential role to confer eye protection from disease states particularly the diseases affecting the retina. Here, we summarize the Hsp function in normal retina, and their involvement in the pathogenesis of certain retinal diseases such cancer, glaucomatous retina, retinitis pigmentosa, and retinal neurodegeneration, as well as the age-related macular degeneration. This information would provide a better understanding of Hsp function and their involvement in ocular disease pathogenesis that could be a target for therapeutic purposes.
Platelets are essential for primary hemostasis and repair of the endothelium, but they also play a key role in the development of acute coronary syndromes and they contribute to cerebrovascular events [1, 2]. In humans, billions of platelets are produced by megakaryocytes and released into the blood stream every day. If they are not consumed in the hemostatic process, these platelets circulate for approximately 10 days before being destroyed by phagocytic cells in the spleen and the liver. While in circulation, platelets remain in a resting state even when exposed to high shear stress or small amounts of soluble agonists generated in the intact vasculature. Vascular injury, however, leads to exposure of platelets to strong agonists, rapid integrin activation, and thrombus formation.
Smad ubiquitin regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that regulates transforming growth factor β (TGF-β)/Smad signaling and is implicated in a wide range of cellular responses. However, the exact mechanism whereby Smurf2 controls TGF-β-induced signaling pathways remains unknown. Here, we identified the relationship between the alternate TGF-β signaling pathways: TGF-β/PI3K/Akt/β-catenin and TGF-β/Smad2/3/FoxO1/PUMA and Smurf2. The results showed that TGF-β promoted proliferation, invasion, and migration of human pancreatic carcinoma (PANC-1) cells through the PI3K/Akt/β-catenin pathway. Inhibiting the PI3K/Akt signal transformed the TGF-β-induced cell response from promoting proliferation to Smad2/3/FoxO1/PUMA-mediated apoptosis. The activation of Akt inhibited the phosphorylation/activation of Smad3 and promoted the phosphorylation/inactivation of FoxO1, inhibiting the nuclear translocation of both Smad3 and FoxO1 and inhibiting the expression of PUMA, a key apoptotic mediator. However, downregulation of Smurf2 in PANC-1 cells removed Akt-mediated suppression of Smad3 and FoxO1, allowing TGF-β-induced phosphorylation/activation of Smad2/3, dephosphorylation/activation of FoxO1, nuclear translocation of both factors, and activation of PUMA-mediated apoptosis. Downregulation of Smurf2 also decreased invasion and migration in TGF-β-induced PANC-1 cells. The in vivo experiments also revealed that downregulation of Smurf2 delayed the growth of xenograft tumors originating from PANC-1 cells especially when treated with TGF-β. Taken together, these results indicate that expression of Smurf2 plays a central role in the determination and activation/inhibition of particular cellular pathways and the ultimate fate of cells induced by TGF-β. An increased understanding of the intricacies of the TGF-β signaling pathway may provide a new anti-cancer therapeutic target.
A single microRNA (miRNA) can regulate expression of multiple proteins, and expression of an individual protein may be controlled by numerous miRNAs. This regulatory pattern strongly suggests that synergistic effects of miRNAs play critical roles in regulating biological processes. miR-9 and miR-124, two of the most abundant miRNAs in the mammalian nervous system, have important functions in neuronal development. In this study, we identified the small GTP-binding protein Rap2a as a common target of both miR-9 and miR-124. miR-9 and miR-124 together, but neither miRNA alone, strongly suppressed Rap2a, thereby promoting neuronal differentiation of neural stem cells (NSCs) and dendritic branching of differentiated neurons. Rap2a also diminished the dendritic complexity of mature neurons by decreasing the levels of pAKT and pGSK3β. Our results reveal a novel pathway in which miR-9 and miR-124 synergistically repress expression of Rap2a to sustain homeostatic dendritic complexity during neuronal development and maturation.
The Rap proteins comprise a subfamily of the Ras-like small G-proteins, most closely related to Ras, which were originally found to antagonize Ras-induced cell transformation. Rap transduces extracellular stimuli to a variety of different processes, amongst others cell-matrix adhesion, cell-cell adhesion, and actin dynamics. Here, we present an overview of the mechanisms that regulate activation, including guanine nucleotide exchange factors and GTPase activating proteins responsible for the regulation. We discuss the various biological functions that are controlled by Rap proteins and the molecular mechanism used by Rap1 to induce these responses.
Background:
Hippocampal neurons in the brain polarize to form multiple dendrites and one long axon. The formation of central synapses remains poorly understood. Although several of the intracellular proteins involved in the clustering of central neurotransmitter receptors and ion channels have been identified, the signals involved in pre- and postsynaptic differentiation remain elusive. Synaptotagmin1 is an abundant and important presynaptic vesicle protein that binds Ca(2+) (J Biol Chem 277:7629-7632, 2002) in regulation of synaptic vesicle exocytosis at the synapse. Synapse consists of the formation of synaptic connections and requires precise coordination of Synaptotagmin1. It was reported Synaptotagmin1 plays an important roles in the formation of axonal filopodia and branches in chicken forebrain neurons (Dev Neurobiol 73:27-44, 2013). To determine if Synaptotagmin1 could have a role in formation of axon in hippocampal neurons, we investigated the effects of Synaptotagmin1 overexpression and knockdown using the shRNA on the growth and branching of the axons of primary hippocampal neurons. We showed that overexpression of Synaptotagmin1 leads to abnormal multiple axon formation in cultured rat hippocampal neurons.
Results:
We first examined the effects of Synaptotagmin1 on the numbers of axon and dendrites. We found that the overexpression of Synaptotagmin1 led to the formation of multiple axons and induced an increase in the number of endogenous postsynaptic protein Homer1c clusters in cultured hippocampal neurons. Endogenous initial segment of axon was detected with anti-sodium channel (anti-NaCh) antibody and with anti-Tau1 (J Neurosci 24: 4605-4613, 2004). The endogenous initial segment of axon was stained with anti-NaCh antibodies and with anti-Tau1 antibodies. Then the numbers of prominence dyed positive were counted as axon. We attempted to specifically knockdown the endogenous Synaptotagmin1 with small hairpin RNAs (shRNAs). To further dissect the functions of endogenous Synaptotagmin1 in neuronal polarity, we used the shRNA of Synaptotagmin1 that specifically blocks the existence of endogenous Synaptotagmin1. When the shRNA of Synaptotagmin1 was introduced to the cells, the number of axons and dendrites did not change.
Conclusions:
These results indicate that the accumulation of Synaptotagmin1 may play an important role in axon/dendrite differentiation.
The numerous biological functions of Ras superfamily small GTPases are highly dependent upon specific posttranslational modifications that guide their subcellular localization and interaction with regulators and effectors. Canonical modifications of their carboxyl termini include prenylation by farnesyl or geranylgeranyl isoprenoid lipids (Ras, Rho, Rab families). These serve as important components of their membrane targeting motifs and promote membrane binding, analogously to the cotranslational amino-terminal myristoylation of Arf family proteins. Reversible carboxymethylation of the prenylated cysteines and reversible acylation by one or more nearby palmitates promote dynamic membrane interactions to complement the permanent lipid modifications. Small GTPases are also regulated in both normal and disease states by several dynamic non-lipid posttranslational modifications. For example, many Ras and Rho family members are phosphorylated in an isoform-specific manner, largely by a select group of serine/threonine kinases such as protein kinase Cα or protein kinase A. Such phosphorylation events, as well as other modifications such as nitrosylation, mono-and di-ubiquitination, peptidyl-prolyl isomerization, acetylation, and oxidation, typically alter small GTPase location and/or interaction with regulatory molecules. By contrast, several distinct E3 ligases posttranslationally regulate small GTPase abundance and function at distinct cellular sites by promoting polyubiquitination and subsequent proteasomal degradation. Finally, numerous pathogenic bacterial toxins disrupt or enhance small GTPase function by a wide variety of posttranslational modifications including ADP ribosylation for which the Arf proteins are named. Here we summarize the rapidly evolving understanding of this fascinating area of small G protein regulation.
Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination.
SIGNIFICANCE STATEMENT:
This study identifies the guanine exchange factor responsible for Rap1b activation during neuronal polarization and provides an alternate explanation for cAMP-dependent acquisition of neuronal polarity.
Neurons are among the most highly polarized cells in our body and the formation of molecularly and functionally distinct axon and dendrites underlies the proper flow of information transfer in the nervous system. This chapter reviews some of the cellular and molecular mechanisms underlying neuronal polarization in vivo focusing, in particular, on the complex interaction between extracellular cues and intracellular signaling mediating axon and dendrite formation.
The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer's disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.Cell Death and Differentiation advance online publication, 12 December 2014; doi:10.1038/cdd.2014.195.
The distinctive polarized morphology of neuronal cells is essential for the proper wiring of the nervous system. The rodent hippocampal neuron culture established about three decades ago has provided an amenable in vitro system to uncover the molecular mechanisms underlying neuronal polarization, a process relying on highly regulated cytoskeletal dynamics, membrane traffic and localized protein degradation. More recent research in vivo has highlighted the importance of the extracellular environment and cell-cell interactions in neuronal polarity. Here, I will review some key signaling pathways regulating neuronal polarization and provide some insights on the complexity of this process gained from in vivo studies.
VISA (also known as MAVS, Cardif, IPS-1) is the essential adaptor protein for virus-induced activation of IFN regulatory factors 3 and 7 and production of type I IFNs. Understanding the regulatory mechanisms for VISA will provide detailed insights into the positive or negative regulation of innate immune responses. In this study, we identified Smad ubiquitin regulatory factor (Smurf) 2, one of the Smad ubiquitin regulator factor proteins, as an important negative regulator of virus-triggered type I IFN signaling, which targets at the VISA level. Overexpression of Smurf2 inhibits virus-induced IFN-β and IFN-stimulated response element activation. The E3 ligase defective mutant Smurf2/C716A loses the ability to suppress virus-induced type I IFN signaling, suggesting that the negative regulation is dependent on the ubiquitin E3 ligase activity of Smurf2. Further studies demonstrated that Smurf2 interacted with VISA and targeted VISA for K48-linked ubiquitination, which promoted the degradation of VISA. Consistently, knockout or knockdown of Smurf2 expression therefore promoted antiviral signaling, which was correlated with the increase in protein stability of VISA. Our findings suggest that Smurf2 is an important nonredundant negative regulator of virus-triggered type I IFN signaling by targeting VISA for K48-linked ubiquitination and degradation.
Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.
Glioblastoma multiforme (GBM) is the most malignant and frequent brain tumor, with an aggressive growth pattern and poor prognosis despite best treatment modalities. Although chemotherapy with temozolomide (TMZ) may restrain tumor growth for some months, TMZ resistance is also common and accounts for many treatment failures. Research into microRNA's role in GBM has shown that microRNAs play a key regulatory role in the GBM, making it a potential therapeutic target. In this study, we demonstrated that the lower expression of miR-181a/b/c/d subunits contributes to astrocytoma tumorigenesis, and their overexpression could inhibit the invasive proliferation of glioblastoma cells by targeting Rap1B-mediated cytoskeleton remodeling and related molecular (Cdc42, RhoA and N-cadherin) changes, suggesting that miR-181 was a critical regulator and might be an important target for glioblastoma treatment. TMZ as a standard chemotherapeutic agent for GBM inhibited the Rap1B expression and actin cytoskeleton remodeling to exert its cell killing by upregulating miR-181a/b/c/d subunits; conversely, each miR-181a/b/c/d subunit enhanced the chemosensitivity of TMZ in glioblastoma cells.
The Ras-like superfamily of low molecular weight GTPases is made of five major families (Arf/Sar, Rab, Ran, Ras, and Rho), highly conserved across evolution. This is in keeping with their roles in basic cellular functions (endo/exocytosis, vesicular trafficking, nucleocytoplasmic trafficking, cell signaling, proliferation and apoptosis, gene regulation, F-actin dynamics), whose alterations are associated with various types of diseases, in particular cancer, neurodegenerative, cardiovascular, and infectious diseases. For these reasons, Ras-like pathways are of great potential in therapeutics and identifying inhibitors that decrease signaling activity is under intense research.Along this line, guanine exchange factors (GEFs) represent attractive targets. GEFs are proteins that promote the active GTP-bound state of GTPases and represent the major entry points whereby extracellular cues are converted into Ras-like signaling. We previously developed the yeast exchange assay (YEA), an experimental setup in the yeast in which activity of a mammalian GEF can be monitored by auxotrophy and color reporter genes. This assay was further engineered for medium-throughput screening of GEF inhibitors, which can readily select for cell-active and specific compounds. We report here on the successful identification of inhibitors against Dbl and CZH/DOCK-family members, GEFs for Rho GTPases, and on the experimental setup to screen for inhibitors of GEFs of the Arf family.We also discuss on inhibitors developed using virtual screening (VS), which target the GEF/GTPase interface with high efficacy and specificity. We propose that using VS and YEA in combination may represent a method of choice for identifying specific and cell-active GEF inhibitors.
Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.
We and others have previously shown that the neuropeptide galanin modulates neurite outgrowth from adult sensory neurons via activation of the second galanin receptor; however, the intracellular signalling pathways that mediate this neuritogenic effect have yet to be elucidated. Here, we demonstrate that galanin decreases the activation state in adult sensory neurons and PC12 cells of Rho and Cdc42 GTPases, both known regulators of filopodial and growth cone motility. Consistent with this, activated levels of Rho and Cdc42 levels are increased in the dorsal root ganglion of adult galanin knockout animals compared with wildtype controls. Furthermore, galanin markedly increases the activation state of cofilin, a downstream effector of many of the small GTPases, in the cell bodies and growth cones of sensory neurons and in PC12 cells. We also demonstrate a reduction in the activation of cofilin, and alteration in growth cone motility, in cultured galanin knockout neurons compared with wildtype controls. These data provide the first evidence that galanin regulates the Rho family of GTPases and cofilin to stimulate growth cone dynamics and neurite outgrowth in sensory neurons. These findings have important therapeutic implications for the treatment of peripheral sensory neuropathies.
Galanin plays a key role in neurite outgrowth from adult sensory neurons via activation of the second galanin receptor (GalR2). Our results demonstrate the galanin decreases the activation state of Rho and Cdc42 and markedly increases the activation of cofilin. These changes lead to alterations in growth cone motility. These findings have important implications for the treatment of peripheral sensory neuropathies.
1,2,3,4-Tetrahydro-6- and -7-methoxy-4-oxo-1-(p-tolylsulfonyl)quinolines 3 (R = tosyl, X = 6- and 7-OMe) and 1-ethoxycarbonylmethyl-1,2,3,4-tetrahydro-7-methoxy-4-oxoquinoline 3 (R = CH2CO2Et, X = 7-OMe) have been ring-expanded in two steps to 2,3,4,5-tetrahydro-7- and -8-methoxy-4-oxo-1-(p-tolylsulfonyl)-1H-1-benzazepines 2 (R = tosyl, X = 7- and -8-OMe) and 1-ethoxycarbonylmethyl-2,3,4,5-tetrahydro-8-methoxy-4-oxo-1H-1-benzazepine 2 (R = CH2CO2Et, X = 8-OMe). Reduction of the oximes gives 4-amino-2,3,4,5-tetrahydro-7-methoxy-1-(p-tolylsulfonyl)-1H-1-benzazepine 1 (R = tosyl, X = 7-OMe), 4-amino-2,3,4,5-tetrahydro-8-methoxy-1H-1-benzazepine 1 (R = H, X = 8-OMe) and 4-amino-1-ethoxycarbonylmethyl-2,3,4,5-tetrahydro-8-methoxy-1H-1-benzazepine 1 (R = CH2CO2Et, X = 8-OMe). From these, several N-substituted and N,N-disubstituted compounds have been obtained and 3-amino-2,3,4,5-tetrahydro-1-(p-tolylsulfonyl)-1H-1-benzazepine 12 (X = NH2, Y = H) has been made by similar means. Two routes are described to 2,3,4,5-tetrahydro-8-methoxy-5-oxo-1-(p-tolylsulfonyl)-1H-1-benzazepine 13 (R1 = tosyl, R2 = H) which is converted to 2,3,4,5-tetrahydro-8-methoxy-4-methoxyimino-5-oxo-1-(p-tolylsulfonyl)-1H-1-benzazepine 17 (R = Me) and thence to 5-[2-(ethoxycarbonyl)ethynyl]-2,3,4,5-tetrahydro-5-hydroxy-8-methoxy-4-methoxyimino-1-(p-tolylsulfonyl)-1H-1-benzazepine 18 (R = CCCO2Et) and 5-[2-(ethoxycarbonyl)ethyl]-2,3,4,5-tetrahydro-5-hydroxy-8-methoxy-4-methoxyimino-1-(p-tolylsulfonyl)-1H-benzazepine 18 [R = (CH2)2CO2Et]. Reduction of oximino ketone 17 (R = H) in two steps gives both cis- and trans-4-acetamido-2,3,4,5-tetrahydro-5-hydroxy-8-methoxy-1-(p-tolylsulfonyl)-1H-1-benzazepines 22 and 21 which are separately deacetylated and cyclised with ethyl chloroacetate to cis- and trans-2,3,4,4a,5,6,7,11b-octahydro-9-methoxy-3-oxo-7-(p-tolylsulfonyl)[1,4]oxazino[3,2-d][1]benzazepine 26 and 25. By similar methodology cis- and trans-2,3,4,5-tetrahydro-5-hydroxy-8-methoxy-4-propionamido-1-(p-tolylsulfonyl)-1H-1-benzazepines 28 and 27 have been obtained, separated and the latter reduced to trans-2,3,4,5-tetrahydro-5-hydroxy-8-methoxy-4-(n-propylamino)-1-(p-tolylsulfonyl)-1H-1-benzazepine 29. In three steps the latter is converted to trans-2,3,4,4a,5,6,7,11b-octahydro-9-methoxy-4-(n-propyl)-7-(p-tolylsulfonyl)[1,4]oxazino[3,2-d][1]benzazepine 33.
Smad ubiquitin regulatory factors (Smurfs) are HECT-domain ubiquitin E3 ligases that regulate diverse cellular processes,
including normal and tumor cell migration. However, the underlying mechanism of the Smurfs' role in cell migration is not
fully understood. Here we show that Smurf1 induces ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4)
at K190. Using the K190R mutant of TRAF4, we demonstrate that Smurf1-induced ubiquitination is required for proper localization
of TRAF4 to tight junctions in confluent epithelial cells. We further show that TRAF4 is essential for the migration of both
normal mammary epithelial and breast cancer cells. The ability of TRAF4 to promote cell migration is also dependent on Smurf1-mediated
ubiquitination, which is associated with Rac1 activation by TRAF4. These results reveal a new regulatory circuit for cell
migration, consisting of Smurf1-mediated ubiquitination of TRAF4 and Rac1 activation.
The orphan nuclear receptor Nur77 regulates diverse cellular activities, including cell proliferation, differentiation and apoptosis. The c-Jun N-terminal kinase (JNK) have a dual role in controlling the function of Nur77. While JNK-mediated phosphorylation of Nur77 positively regulates its translocation to the mitochondria to induce apoptosis, it negatively regulates the stability of Nur77. The underlying mechanism for the dual role of JNK in regulating Nur77, however, is unclear. Here, we report that E3 ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) prevents Nur77 degradation through mediating its unconventional ubiquitination, thereby mitigating the JNK-mediated downregulating effect, which leads to Nur77 accumulation and subsequent translocation to mitochondria to trigger apoptosis. In this process, protein kinase A (PKA)-mediated phosphorylation of Smurf1 at Thr306 is a prerequisite step. Accordingly, cyclic AMP/PKA signaling switches the fate of Nur77 from degradation to triggering apoptosis in chemotherapy drug cisplatin-treated cells. Hence, our study revealed a novel mechanism, by which PKA/Smurf1 antagonizes the downregulating effect of JNK on Nur77, leading to the accumulation of Nur77 for apoptosis induction triggered by cisplatin.Oncogene advance online publication, 15 April 2013; doi:10.1038/onc.2013.116.
Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor (ESFT) cell lines was mediated by Frizzled3, Dishevelled (Dvl) and c-Jun N-terminal kinase (Endo et al. MCB, 2008). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1 delta, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer and Rubin JCB, 2011). However, the functional relevance of Dvl phophorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical protein kinase C zeta (PKCζ), but the role of Dvl phosphorylation was not evaluated (Zhang et al. Nature Cell Biol, 2007). Here we report that ESFT cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of p-PKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. While FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (submitted manuscript). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.
When the CNS is injured, damaged axons do not regenerate. This failure is due in part to the growth-inhibitory environment that forms at the injury site. Myelin-associated molecules, repulsive axon guidance molecules, and extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs) found within the glial scar inhibit axon regeneration but the intracellular signaling mechanisms triggered by these diverse molecules remain largely unknown. Here we provide biochemical and functional evidence that atypical protein kinase C (PKCζ) and polarity (Par) complex proteins mediate axon growth inhibition. Treatment of postnatal rat neurons in vitro with the NG2 CSPG, a major component of the glial scar, activates PKCζ, and this activation is both necessary and sufficient to inhibit axonal growth. NG2 treatment also activates Cdc42, increases the association of Par6 with PKCζ, and leads to a Par3-dependent activation of Rac1. Transfection of neurons with kinase-dead forms of PKCζ, dominant-negative forms of Cdc42, or mutant forms of Par6 that do not bind to Cdc42 prevent NG2-induced growth inhibition. Similarly, transfection with either a phosphomutant Par3 (S824A) or dominant-negative Rac1 prevent inhibition, whereas expression of constitutively active Rac1 inhibits axon growth on control surfaces. These results suggest a model in which NG2 binding to neurons activates PKCζ and modifies Par complex function. They also identify the Par complex as a novel therapeutic target for promoting axon regeneration after CNS injury.
Polarization of mammalian neurons with a specified axon requires precise regulation of microtubule and actin dynamics in the developing neurites. Here we show that mammalian partition defective 3 (mPar3), a key component of the Par polarity complex that regulates the polarization of many cell types including neurons, directly regulates microtubule stability and organization. The N-terminal portion of mPar3 exhibits strong microtubule binding, bundling, and stabilization activity, which can be suppressed by its C-terminal portion via an intramolecular interaction. Interestingly, the intermolecular oligomerization of mPar3 is able to relieve the intramolecular interaction and thereby promote microtubule bundling and stabilization. Furthermore, disruption of this microtubule regulatory activity of mPar3 impairs its function in axon specification. Together, these results demonstrate a role for mPar3 in directly regulating microtubule organization that is crucial for neuronal polarization.
Viral and bacterial infection activate several transcription factors including NF-κB and IRF3, which collaborate to induce type I interferons (IFNs). However, the mechanisms involved in terminating the production of type I IFNs are not well defined. Here we identified Smurf2 as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of Smurf2 expression by small interfering RNA resulted in augmented activation of IRF3 and enhanced expression of IFN-β, whereas overexpression of Smurf2 had opposite effects. As an HECT domain E3 ligase, Smurf2 interacted with activated IRF3 directly and catalyzed K48-linked polyubiquitination of phosphorylated IRF3. These findings suggest that Smurf2 is a negative regulator for TLR3/4- and RIG-I-mediated IFN-β production by targeting phosphorylated IRF3 for ubiquitination and subsequent proteasome-mediated degradation.
Smad ubiquitin regulatory factors (Smurfs) belong to the HECT- family of E3 ubiquitin ligases and comprise mainly of two members, Smurf1 and Smurf2. Initially, Smurfs have been implicated in determining the competence of cells to respond to TGF- β/BMP signaling pathway. Nevertheless, the intrinsic catalytic activity has extended the repertoire of Smurf substrates beyond the TGF-β/BMP super family expanding its realm further to epigenetic modifications of histones governing the chromatin landscape. Through regulation of a large number of proteins in multiple cellular compartments, Smurfs regulate diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and metastasis. As the genomic ablation of Smurfs leads to global changes in histone modifications and predisposition to a wide spectrum of tumors, Smurfs are also considered to have a novel tumor suppressor function. This review focuses on regulation network and biological functions of Smurfs in connection with its role in cancer progression. By providing a portrait of their protein targets, we intend to link the substrate specificity of Smurfs with their contribution to tumorigenesis. Since the regulation and biological functions of Smurfs are quite complex, understanding the oncogenic potential of these E3 ubiquitin ligases may facilitate the development of mechanism-based drugs in cancer treatment.
Differentiation of axons and dendrites is a critical step in neuronal development. Here we review the evidence that axon/dendrite formation during neuronal polarization depends on the intrinsic cytoplasmic asymmetry inherited by the postmitotic neuron, the exposure of the neuron to extracellular chemical factors, and the action of anisotropic mechanical forces imposed by the environment. To better delineate the functions of early signals among a myriad of cellular components that were shown to influence axon/dendrite formation, we discuss their functions by distinguishing their roles as determinants, mediators, or modulators and consider selective degradation of these components as a potential mechanism for axon/dendrite polarization. Finally, we examine whether these early events of axon/dendrite formation involve local autocatalytic activation and long-range inhibition, as postulated by Alan Turing for the morphogenesis of patterned biological structure.
Mechanisms determining neuronal polarity have been most extensively studied using dissociated hippocampal neurons which develop
in a very stereotypic manner in the absence of differential extrinsic cues. Polarity is established in two major steps: first,
an initial deformation of the spherical cell gives rise to the first neurite, and later, after multiple neurites grew from
the sphere, one neurite will be selected for fast axonal outgrowth. Both steps are under the tight control of cytoskeletal
rearrangements; sub-membranous local actin remodelling will support the disruption of the spherical architecture to thus allow
the formation of the first neurite and from this, later on, rapid growth. Axonal growth from a multipolar neuron at later
stages has been shown to be supported by local actin destabilization and that could be achieved either by intrinsic or extrinsic
cues, demonstrating that polarity establishment is a complex endogenously controlled remodelling process, under active control
by extrinsic factors.
KeywordsHippocampal neurons-Permissive-Instructive-Growth cone-Minor neurite-Dendrite-Axon
The remodeling of synapses is a fundamental mechanism for information storage and processing in the brain. During brain development, and in response to learningrelated activity, synapses undergo remarkable structural changes, including growth, shrinkage, and elimination. Such structural plasticity provides a physical basis for enduring changes in neural circuits that are mediated by alterations in the molecular composition of the synapse. Indeed, stabilization or removal of neurotransmitter receptors, scaffold proteins, and signaling molecules from the synapse has been proposed to account for long-term changes in synaptic strength. Patterns of molecular changes in large sets of synaptic proteins, which ultimately encode the history of activity at the synapse, represent a level of complexity that we are only now beginning to understand. Long-lasting changes in the molecular content of synapses arise by two general mechanisms: the incorporation of new proteins and the selective removal of existing synaptic proteins. For much of the past two decades, the prevailing model for enduring changes in synapse function and structure has been stimulus-dependent gene expression and protein synthesis. Indeed, substantial evidence indicates that transcriptional events are critical for long-term activity-dependent plasticity (135, 184). In addition, local translation of mRNAs is thought to orchestrate long-lasting forms of learning-related synaptic plasticity (269). On the other hand, considerably less attention has been given to the contribution of protein turnover to long-term structural and functional changes at synapses. What controls the turnover and replacement of synaptic proteins? For most cellular proteins, the major pathway of degradation occurs by ubiquitin conjugation and subsequent targeting of ubiquitin-conjugated proteins to the proteasome. Here we will discuss the role of the ubiquitin-proteasome system (UPS) in the structural and functional regulation of synapses. In addition to targeting proteins to the proteasome, post-translational modification by ubiquitination can trigger the assembly of modular protein complexes that recognize ubiquitin moieties in a manner similar to protein phosphorylation (146). In this case, ubiquitination can regulate protein function and protein trafficking (198). We will further discuss recent work revealing ubiquitindependent protein modification in synaptic signaling. Far from being immutable structures, synapses contain diverse interconnected protein networks within which reside the molecular traces of experience and memory. Central to how synapses change and how synaptic molecules store information is how neurons regulate protein degradation and replacement at the synapse. Our focus in this chapter will be to summarize current knowledge of ubiquitination and protein degradation at synapses, a topic that promises to provide a new conceptual framework for understanding both the plasticity and persistence of neural circuits. © 2008 Springer Science+Business Media, LLC. All rights reserved.
Members of the Ras superfamily of small guanosine triphosphatases (GTPases) function as key nodes within signaling networks in a remarkable range of cellular processes, including cell proliferation, differentiation, growth, cell-cell adhesion and apoptosis. We recently described a novel role for the Ras-like small GTPases Rap1 and Ral in regulating cortical polarity and spindle orientation during asymmetric neuroblast division in Drosophila. The participation of these proteins in promoting cell polarization seems to be a common theme throughout evolution.
The E3 ubiquitin ligase Smurf2 mediates ubiquitination and degradation of several protein targets involved in tumorigenesis and induces senescence in human cells. However, the functional role of Smurf2 in tumorigenesis has not been fully evaluated. In this study, we generated a mouse model of Smurf2 deficiency to characterize the function of this E3 ligase in tumorigenesis. Smurf2 deficiency attenuated p16 expression and impaired the senescence response of primary mouse embryonic fibroblasts. In support of a functional role in controlling cancer, Smurf2 deficiency increased the susceptibility of mice to spontaneous tumorigenesis, most notably B-cell lymphoma. At a premalignant stage of tumorigenesis, we documented a defective senescence response in the spleens of Smurf2-deficient mice, consistent with a mechanistic link between impaired senescence regulation and increased tumorigenesis. Taken together, our findings offer the genetic evidence of an important tumor suppressor function for Smurf2.
Transforming growth factor β (TGFβ) regulates many physiological processes and requires control mechanisms to safeguard proper
and timely action. We have previously described how negative regulation of TGFβ signaling is controlled by the serine/threonine
kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFβ type I receptor and with the inhibitory Smad7 and
down-regulates the type I receptor. We now demonstrate that TGFβ induces SIK1 levels via a direct transcriptional mechanism
that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the
kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We
also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFβ that leads to epithelial growth
arrest. In conclusion, TGFβ induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally
interlink to control the activity of this pathway.
Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.
In addition to allelic mutations, cancers are known to harbor alterations in their chromatin landscape. Here we show that genomic ablation of Smad ubiquitin regulatory factor 2 (Smurf2), a HECT-domain E3 ubiquitin ligase, results in dysregulation of both the DNA damage response and genomic stability, culminating in increased susceptibility to various types of cancers in aged mice. We show that Smurf2 regulates the monoubiquitination of histone H2B as well as the trimethylation of histone H3 at Lys4 and Lys79 by targeting ring finger protein 20 (RNF20) for proteasomal degradation in both mouse and human cells. We also show that Smurf2 and RNF20 are colocalized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus. Thus, Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.
The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.
By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition, and in their synaptic polarity (Bartlett and Banker, 1984a, b; Caceres et al., 1984). We examined the events during the first week in culture that lead to the establishment of this characteristic form. Hippocampal cells were obtained from 18 d fetal rats, plated onto polylysine-treated coverslips, and maintained in a serum-free medium. The development of individual cells was followed by sequential photography at daily intervals until both axons and dendrites had been established; identification of the processes was confirmed by immunostaining for MAP2, a dendritic marker. Time-lapse video recording was used to follow the early stages of process formation. Hippocampal neurons acquired their characteristic form by a stereotyped sequence of developmental events. The cells first established several, apparently identical, short processes. After several hours, one of the short processes began to grow very rapidly; it became the axon. The remaining processes began to elongate a few days later and grew at a much slower rate. They became the cell's dendrites. Neurons that arose following mitosis in culture underwent this same sequence of developmental events. In a few instances, 2 processes from a cell exhibited the rapid growth typical of axons, but only one maintained this growth; the other retracted and became a dendrite. Axons branched primarily by the formation of collaterals, not by bifurcation of growth cones. As judged by light microscopy, processes are not specified as axons or dendrites when they arise. The first manifestation of neuronal polarity is the acquisition of axonal characteristics by one of the initial processes; subsequently the remaining processes become dendrites.
By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition, and in their synaptic polarity (Bartlett and Banker, 1984a, b; Caceres et al., 1984). We examined the events during the first week in culture that lead to the establishment of this characteristic form. Hippocampal cells were obtained from 18 d fetal rats, plated onto polylysine-treated coverslips, and maintained in a serum-free medium. The development of individual cells was followed by sequential photography at daily intervals until both axons and dendrites had been established; identification of the processes was confirmed by immunostaining for MAP2, a dendritic marker. Time-lapse video recording was used to follow the early stages of process formation. Hippocampal neurons acquired their characteristic form by a stereotyped sequence of developmental events. The cells first established several, apparently identical, short processes. After several hours, one of the short processes began to grow very rapidly; it became the axon. The remaining processes began to elongate a few days later and grew at a much slower rate. They became the cell's dendrites. Neurons that arose following mitosis in culture underwent this same sequence of developmental events. In a few instances, 2 processes from a cell exhibited the rapid growth typical of axons, but only one maintained this growth; the other retracted and became a dendrite. Axons branched primarily by the formation of collaterals, not by bifurcation of growth cones. As judged by light microscopy, processes are not specified as axons or dendrites when they arise. The first manifestation of neuronal polarity is the acquisition of axonal characteristics by one of the initial processes; subsequently the remaining processes become dendrites.
Members of the Ras superfamily of small molecular weight GTPases play diverse and critical roles in mediating cellular responses
to extracellular stimuli, including mitogenesis, cytoskeletal maintenance and rearrangement, and integrin activation. In T
lymphocytes, biochemical and genetic evidence demonstrate that Ras plays an essential role in coupling T cell receptor ligation
to signaling cascades required for T cell proliferation and development. Recent observations that C3G, a guanine nucleotide
exchange factor specific for the Ras-related GTPase Rap1, is recruited into tyrosine-phosphorylated protein signaling complexes
in activated T cells have suggested that Rap1 may also play a role in T cell activation. Utilizing a recently developed technique
for detection of endogenous, GTP-bound Rap1, we have found that Rap1, but not Rap2, is transiently activated following T cell
receptor stimulation of normal human T lymphocytes. Increases in intracellular calcium is both necessary and sufficient to
induce Rap1 activation. Remarkably, costimulation of T cells with mitogenic anti-CD28 antibody completely abolished T cell
receptor-dependent activation of Rap1. This report demonstrates a potential role for Rap1 in T cell receptor signaling and
suggests inactivation of Rap1 as a candidate target of CD28-dependent costimulatory signals required for T cell antigen responsiveness.
Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.
The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals.
In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.
RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.
Calcium is a universal intracellular signal that is responsible for controlling a plethora of cellular processes. Understanding how such a simple ion can regulate so many diverse cellular processes is a key goal of calcium- and cell-biologists. One molecule that is sensitive to changes in intracellular calcium levels is Ras. This small GTPase operates as a binary molecular switch, and regulates cell proliferation and differentiation. Here, we focus on examining the link between calcium and Ras signalling and, in particular, we speculate as to how the complexity of calcium signalling could regulate Ras activity.
Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.
cAMP is involved in a wide variety of cellular processes that were thought to be mediated by protein kinase A (PKA). However, cAMP also directly regulates Epac1 and Epac2, guanine nucleotide-exchange factors (GEFs) for the small GTPases Rap1 and Rap2 (refs 2,3). Unfortunately, there is an absence of tools to discriminate between PKA- and Epac-mediated effects. Therefore, through rational drug design we have developed a novel cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), which activates Epac, but not PKA, both in vitro and in vivo. Using this analogue, we tested the widespread model that Rap1 mediates cAMP-induced regulation of the extracellular signal-regulated kinase (ERK). However, both in cell lines in which cAMP inhibits growth-factor-induced ERK activation and in which cAMP activates ERK, 8CPT-2Me-cAMP did not affect ERK activity. Moreover, in cell lines in which cAMP activates ERK, inhibition of PKA and Ras, but not Rap1, abolished cAMP-mediated ERK activation. We conclude that cAMP-induced regulation of ERK and activation of Rap1 are independent processes.
The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such
as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity
and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein
kinase C zeta (PKCζ), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where
it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia
to prevent RhoA signaling during dynamic membrane movements.
The anaphase-promoting complex (APC) is highly expressed in postmitotic neurons, but its function in the nervous system was
previously unknown. We report that the inhibition of Cdh1-APC in primary neurons specifically enhanced axonal growth. Cdh1
knockdown in cerebellar slice overlay assays and in the developing rat cerebellum in vivo revealed cell-autonomous abnormalities
in layer-specific growth of granule neuron axons and parallel fiber patterning. Cdh1 RNA interference in neurons was also
found to override the inhibitory influence of myelin on axonal growth. Thus, Cdh1-APC appears to play a role in regulating
axonal growth and patterning in the developing brain that may also limit the growth of injured axons in the adult brain.
Neurons polarize to form elaborate multiple dendrites and one long axon. The establishment and maintenance of axon/dendrite polarity are fundamentally important for neurons. Recent studies have demonstrated that the polarity complex PAR-3-PAR-6-atypical protein kinase C (aPKC) is involved in polarity determination in many tissues and cells. The function of the PAR-3-PAR-6-aPKC protein complex depends on its subcellular localization in polarized cells. PAR-3 accumulates at the tip of growing axons in cultured rat hippocampal neurons, but the molecular mechanism of this localization remains unknown. Here we identify a direct interaction between PAR-3 and KIF3A, a plus-end-directed microtubule motor protein, and show that aPKC can associate with KIF3A through its interaction with PAR-3. The expression of dominant-negative PAR-3 and KIF3A fragments that disrupt PAR-3-KIF3A binding inhibited the accumulation of PAR-3 and aPKC at the tip of the neurites and abolished neuronal polarity. These results suggest that PAR-3 is transported to the distal tip of the axon by KIF3A and that the proper localization of PAR-3 is required to establish neuronal polarity.
The secreted semaphorin 3A (Sema3A) is a member of a large family of proteins that act as guidance signals for axons and dendrites.
While the receptors and signaling pathways that mediate the repulsive effects of semaphorins are beginning to be understood
in some detail, the mechanisms that are responsible for the ability of Sema3A to stimulate the extension of dendrites remain
to be elucidated. Here we show that PC12 cells, a model widely used to study neuronal differentiation, can be used to dissect
this pathway. Sema3A is as effective as nerve growth factor in stimulating the extension of neurites from PC12 cells. We show
that Sema3A is able to regulate gene expression and identify mitochondria as a novel target of Sema3A signaling. Pharmacological
block of mitochondrial reactive oxygen species production abolishes the extension of neurites in response to Sema3A. These
results show that the characterization of transcripts that are regulated by axon guidance signals may help to identify novel
components of their signaling pathways.
Posttranslational modification of cellular proteins by the covalent attachment of ubiquitin regulates protein stability, activity, and localization. Ubiquitination is rapid and reversible and is a potent mechanism for the spatial and temporal control of protein activity. By sculpting the molecular composition of the synapse, this versatile posttranslational modification shapes the pattern, activity, and plasticity of synaptic connections. Synaptic processes regulated by ubiquitination, as well as ubiquitination enzymes and their targets at the synapse, are being identified by genetic, biochemical, and electrophysiological analyses. This work provides tantalizing hints that neuronal activity collaborates with ubiquitination pathways to regulate the structure and function of synapses.
Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.
The transition of cells from an epithelial to a mesenchymal phenotype is a critical event during morphogenesis in multicellular
organisms and underlies the pathology of many diseases, including the invasive phenotype associated with metastatic carcinomas.
Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT). However, the molecular
mechanisms that control the dissolution of tight junctions, an early event in EMT, remain elusive. We demonstrate that Par6,
a regulator of epithelial cell polarity and tight-junction assembly, interacts with TGFβ receptors and is a substrate of the
type II receptor, TβRII. Phosphorylation of Par6 is required for TGFβ-dependent EMT in mammary gland epithelial cells and
controls the interaction of Par6 with the E3 ubiquitin ligase Smurf1. Smurf1, in turn, targets the guanosine triphosphatase
RhoA for degradation, thereby leading to a loss of tight junctions. These studies define how an extracellular cue signals
to the polarity machinery to control epithelial cell morphology.
PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes, including cell migration and chemotaxis. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium and mouse neutrophils. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.
Axon specification triggers the polarization of neurons and requires the localized destabilization of filamentous actin. Here we show that plasma membrane ganglioside sialidase (PMGS) asymmetrically accumulates at the tip of one neurite of the unpolarized rat neuron, inducing actin instability. Suppressing PMGS activity blocks axonal generation, whereas stimulating it accelerates the formation of a single (not several) axon. PMGS induces axon specification by enhancing TrkA activity locally, which triggers phosphatidylinositol-3-kinase (PI3K)- and Rac1-dependent inhibition of RhoA signaling and the consequent actin depolymerization in one neurite only. Thus, spatial restriction of an actin-regulating molecular machinery, in this case a membrane enzymatic activity, before polarization is enough to determine axonal fate.
Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1
and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP),
RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed
GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression
of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP
reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and
regulates the neurite outgrowth.
Neuronal polarization occurs shortly after mitosis. In neurons differentiating in vitro, axon formation follows the segregation of growth-promoting activities to only one of the multiple neurites that form after mitosis. It is unresolved whether such spatial restriction makes use of an intrinsic program, like during C. elegans embryo polarization, or is extrinsic and cue-mediated, as in migratory cells. Here we show that in hippocampal neurons in vitro, the axon consistently arises from the neurite that develops first after mitosis. Centrosomes, the Golgi apparatus and endosomes cluster together close to the area where the first neurite will form, which is in turn opposite from the plane of the last mitotic division. We show that the polarized activities of these organelles are necessary and sufficient for neuronal polarization: (1) polarized microtubule polymerization and membrane transport precedes first neurite formation, (2) neurons with more than one centrosome sprout more than one axon and (3) suppression of centrosome-mediated functions precludes polarization. We conclude that asymmetric centrosome-mediated dynamics in the early post-mitotic stage instruct neuronal polarity, implying that pre-mitotic mechanisms with a role in division orientation may in turn participate in this event.
Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.
Asymmetric distributions of activities of the protein kinases Akt and glycogen synthase kinase 3beta (GSK-3beta) are critical for the formation of neuronal polarity. However, the mechanisms underlying polarized regulation of this pathway remain unclear. In this study, we report that the instability of Akt regulated by the ubiquitin-proteasome system (UPS) is required for neuron polarity. Preferential distribution in the axons was observed for Akt but not for its target GSK-3beta. A photoactivatable GFP fused to Akt revealed the preferential instability of Akt in dendrites. Akt but not p110 or GSK-3beta was ubiquitinated. Suppressing the UPS led to the symmetric distribution of Akt and the formation of multiple axons. These results indicate that local protein degradation mediated by the UPS is important in determining neuronal polarity.
The role of the ubiquitin-dependent proteolysis system in c-Jun breakdown was investigated. Using in vitro experiments and a novel in vivo assay that utilizes molecularly-tagged ubiquitin and c-Jun proteins, it was shown that c-Jun, but not its transforming counterpart, retroviral v-Jun, can be efficiently multiubiquitinated. Consistently, v-Jun has a longer half-life than c-Jun. Mutagenesis experiments indicate that the reason for the escape of v-Jun from multiubiquitination and its resulting stabilization is the deletion of the delta domain, a stretch of 27 amino acids that is present in c-Jun but not in v-Jun. c-Jun sequences containing the delta domain, when transferred to the bacterial beta-galactosidase protein, function as a cis-acting ubiquitination and degradation signal. The correlation between transforming ability and the escape from ubiquitin-dependent degradation described here suggests a novel route to oncogenesis.
Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration.
Axon formation in multipolar neurons is believed to depend on the existence of precise sorting mechanisms for axonal membrane and membrane-associated proteins. Conclusive evidence in living neurons, however, is lacking. In the present study, we use light and video microscopy to address this issue directly. We show that axon formation is preceded by the appearance in one of the multiple neurites of (1) a larger growth cone, (2) a higher amount and greater transport of membrane organelles, (3) polarized delivery of TGN-derived vesicles, (4) a higher concentration of mitochondria and peroxisomes, (5) a higher concentration of a cytosolic protein, and (6) a higher concentration of ribosomes. These results provide evidence for the involvement of bulk cytoplasmic flow as an early determinant of neuronal morphological polarization. Molecular sorting events would later trigger the establishment of functional polarity.
In recent years we have learned a great deal about the molecular mechanisms underlying axonal elongation and navigation and the manner in which extracellular signals modify a growth cone's course of action. Yet, the mechanisms responsible for the earlier events of axonal and dendritic generation are just beginning to be understood. The recent advances in this exciting field highlight the importance of studies of cell migration and axonal elongation for our current understanding of the establishment of neuronal polarity.
Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.
Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.
Growth cones contain mRNAs, translation machinery, and, as we report here, protein degradation machinery. We show that isolated retinal growth cones immediately lose their ability to turn in a chemotropic gradient of netrin-1 or Sema3A when translation is inhibited. Translation inhibition also prevents Sema3A-induced collapse, while LPA-induced collapse is not affected. Inhibition of proteasome function blocks responses to netrin-1 and LPA but does not affect Sema3A responses. We further demonstrate in isolated growth cones that netrin-1 and Sema3A activate translation initiation factors and stimulate a marked rise in protein synthesis within minutes, while netrin-1 and LPA elicit similar rises in ubiquitin-protein conjugates. These results suggest that guidance molecules steer axon growth by triggering rapid local changes in protein levels in growth cones.
Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, considered to be a nonspecific, dead-end process. Although it was known that proteins do turn over, the large extent and high specificity of the process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease. With the multitude of substrates targeted and the myriad processes involved, it is not surprising that aberrations in the pathway are implicated in the pathogenesis of many diseases, certain malignancies, and neurodegeneration among them. Degradation of a protein via the ubiquitin/proteasome pathway involves two successive steps: 1) conjugation of multiple ubiquitin moieties to the substrate and 2) degradation of the tagged protein by the downstream 26S proteasome complex. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation, and major key questions have remained unsolved. Among these are the modes of specific and timed recognition for the degradation of the many substrates and the mechanisms that underlie aberrations in the system that lead to pathogenesis of diseases.
The sprouting of neurites, which will later become axons and dendrites, is an important event in early neuronal differentiation. Studies in living neurons indicate that neuritogenesis begins immediately after neuronal commitment, with the activation of membrane receptors by extracellular cues. These receptors activate intracellular cascades that trigger changes in the actin cytoskeleton, which promote the initial breakdown of symmetry. Then, through the regulation of gene transcription, and of microtubule and membrane dynamics, the newly formed neurite becomes stabilized. A key challenge is to define the molecular machinery that regulates the actin cytoskeleton during initial neurite sprouting. We propose that analysing the molecules involved in actin-dependent mechanisms in non-neuronal systems, such as budding yeast and migrating fibroblasts, could help to uncover the secrets of neuritogenesis.
How a neuron becomes polarized remains an outstanding question. Here, we report that selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity.
Previous work has shown that guidance cues trigger rapid changes in protein dynamics in retinal growth cones: netrin-1 stimulates both protein synthesis and degradation, while Sema3A elicits synthesis, and LPA induces degradation. What signaling pathways are involved? Our studies confirm that p42/44 MAPK mediates netrin-1 responses and further show that inhibiting its activity blocks cue-induced protein synthesis. Unexpectedly, p38 MAPK is also activated by netrin-1 in retinal growth cones and is required for chemotropic responses and translation. Sema3A- and LPA-induced responses, by contrast, require a single MAPK, p42/p44 and p38, respectively. In addition, we report that caspase-3, an apoptotic protease, is rapidly activated by netrin-1 and LPA in a proteasome- and p38-dependent manner and is required for chemotropic responses. These findings suggest that the apoptotic pathway may be used locally to control protein levels in growth cones and that the differential activation of MAPK pathways may underlie cue-directed migration.
Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3beta (GSK-3beta). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway.
The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.
The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.
Neuronal plasticity relies on tightly regulated control of protein levels at synapses. One mechanism to control protein abundance is the ubiquitin-proteasome degradation system. Recent studies have implicated ubiquitin-mediated protein degradation in synaptic development, function, and plasticity, but little is known about the regulatory mechanisms controlling ubiquitylation in neurons. In contrast, ubiquitylation has long been studied as a central regulator of the eukaryotic cell cycle. A critical mediator of cell-cycle transitions, the anaphase-promoting complex/cyclosome (APC/C), is an E3 ubiquitin ligase. Although the APC/C has been detected in several differentiated cell types, a functional role for the complex in postmitotic cells has been elusive. We describe a novel postmitotic role for the APC/C at Drosophila neuromuscular synapses: independent regulation of synaptic growth and synaptic transmission. In neurons, the APC/C controls synaptic size via a downstream effector Liprin-alpha; in muscles, the APC/C regulates synaptic transmission, controlling the concentration of a postsynaptic glutamate receptor.
In developing hippocampal neurons in culture, the evolutionarily conserved polarity complex mPar3/mPar6/aPKC selectively accumulates at the tip of one, and only one, of the immature neurites of a neuron and thus specifies the axon and generates neuronal polarity. How mPar3/mPar6 is enriched at the tip of the nascent axon, but not the dendrites, is not fully understood. Here, we report that mPar3 forms a complex with adenomatous polyposis coli (APC) and kinesin superfamily (KIF) 3A, proteins that move along microtubules. In polarizing hippocampal neurons, APC selectively accumulates at the nascent axon tip and colocalizes with mPar3. Expression of dominant-negative C terminus deletion mutants of APC or ectopic expression of APC leads to dislocalization of mPar3 and defects in axon specification and neuronal polarity. In addition to spatial polarization of APC, the selective inactivation of the GSK-3beta activity at the nascent axon tip is required for mPar3 targeting and polarization and establishing neuronal polarity. These results suggest that mPar3 is polarized in developing neurons through APC- and kinesin-mediated transport to the plus ends of rapidly growing microtubules at the nascent axon tip, a process that involves a spatially regulated GSK-3beta activity.
The investigation of protein function through the inhibition of activity has been critical to our understanding of many normal and abnormal biological processes. Until recently, functional inhibition in biological systems has been induced using a variety of approaches including small molecule antagonists, antibodies, aptamers, ribozymes, antisense oligonucleotides or transcripts, morpholinos, dominant-negative mutants, and knockout transgenic animals. Although all of these approaches have made substantial advances in our understanding of the function of many proteins, a lack of specificity or restricted applicability has limited their utility. Recently, exploitation of the naturally occurring posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), termed RNA interference (RNAi), has gained much favor as an alternative means for analyzing gene function. Aspects of the basic biology of RNAi, its application as a functional genomics tool, and its potential as a therapeutic approach have been extensively reviewed (Hannon and Rossi, 2004; Meister and Tuschl, 2004); however, there has been only limited discussion as to how to design and validate an individual RNAi effector molecule and how to interpret RNAi data overall, particularly with reference to experimentation in mammalian cells. This perspective will aim to consider some of the issues encountered when conducting and interpreting RNAi experiments in mammalian cells.
Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. Here we report a differential distribution of GSK-3beta activity in the axon versus the dendrites. A constitutively active GSK-3beta mutant inhibited axon formation, whereas multiple axons formed from a single neuron when GSK-3beta activity was reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3beta in determining neuronal polarity. Our results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3beta relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3beta inhibitors can be a novel approach to promote generation of new axons after neural injuries.
Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.
The ability of cells to polarize is critical for complex biological activities, such as the organization of the nervous system. Indeed, neurons are among the best examples of a highly differentiated and polarized cell type, typically extending a long thin axon, which is engineered to propagate signals, and several shorter and thicker dendrites, which are designed to receive signal inputs. The transfer of information from a neuron to its target occurs at the synapse, which are composed of specialized pre- and postsynaptic structures. The presynaptic terminal stores vesicles, which upon activation release neurotransmitters into the synaptic space, where they act on postsynaptic receptors. The asymmetric localization of proteins within the axon, dendrite, and synapse is essential for a neuron to establish its functional architecture.
Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated hippocampal neurons are a widely used system to study the development of neuronal differentiation. In this article, we will describe gain-of-function and loss-of-function approaches that allow us to analyze the role of GTPases in neuronal differentiation.
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