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Annexins II, IV, V and VI relocate in response to rises in intracellular calcium in human foreskin fibroblasts
Abstract
Annexins are a family of proteins implicated in a number of cellular processes involving calcium. We studied annexins I, II, IV, V and VI and found that they are all present in human foreskin fibroblasts and, from immunocytochemical studies, have distinct locations in the cell. Only annexin IV and annexin V have unstructured cytoplasmic staining patterns consistent with predominantly cytosolic locations. Annexin VI partially colocalizes with the endoplasmic reticulum. In contrast, annexins I and II are both associated with the plasma membrane with annexin II having a very homogeneous staining compared with the punctate pattern observed for annexin I. Annexins I, IV and V are all present in the nucleus at higher concentrations than in the cytoplasm. Treatment of cells with the calcium ionophore A23187 to raise intracellular calcium, results in relocations of annexin II, IV, V and VI. Intranuclear annexins IV and V relocate to the nuclear membrane whereas the cytosolic pools of these annexins relocate to the plasma membrane. Annexin II relocates to granular structures at the plasma membrane whereas annexin VI relocates to a more homogeneous distribution on the plasma membrane. These results are consistent with an important role for annexins in mediating the calcium signal at the plasma membrane and within the nuclei of fibroblasts.
... In particular, annexin 1 (ANXA1) is known to regulate second-phase insulin secretion (17), and ANXA2 is involved in actin-dependent vesicle transport (18)(19)(20)(21). Annexins respond to increased [Ca 2+ ] i by shuttling between the nucleus and the plasma membrane (22). Cellular localization of annexins is mediated via formation of heterotetrameric complexes through binding to the S100 protein family (15,23). ...
... Loss of ZNF148 restores translocation of annexins to cellular membranes in SC-β cells. Annexins link Ca 2+ signaling and membrane functions as they relocate from within nuclei to the nuclear and plasma membrane in response to elevated [Ca 2+ ] i (15,22). Cellular translocation requires the formation of heterotetrameric complexes with members of the S100 family; these complexes regulate the organization of membranes and vesicles and have been involved in fusion of vesicles with the plasma membrane (24). ...
Insulin secretion from pancreatic β cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell-derived β cells (SC-β cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC-β cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC-β cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human β cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.
... Annexin V is one of the most widespread and abundant annexins. We have demonstrated that annexin V is expressed human osteosarcoma cell line MG-63 at high concentration in the nuclei of fibroblasts [7]. In this study, we investigated the effect of some serum factors on the tyrosine kinase signaling pathways in the nuclear location of annexin V. ...
... Preincubation of the antiserum to annexin V with pure annexin V was able to totally remove immune staining obtained with the polyclonal antiserum. The anti-serum to annexin V has been previously shown to be specific by Western blotting analysis of several cell types including MG-63 cells [7,8]. ...
Calcium-binding proteins play essential roles in the cell. One important class of calcium-binding proteins is the annexin family. This is a family of 13 proteins, which binds to phospholipids in a calcium-dependent manner. Osteosarcoma cell line (MG-63) is a transformed cell that has many characteristics of the differentiated cell, such as a considerable serum dependency in its growth rate. Using specific antibodies against each annexin and immunoflurescence microscopy, the location and relocation of the annexin V was determined by some serum factors. Serum starvation of MG-63 cells increases their doubling time from 24 hours to 4 days. Cells grown in serum contain high levels of annexin V in the cell nucleus whereas in the absence of serum results in loss of nuclear annexin V in about 75% of the cells. Refeeding cells with medium containing 10% serum restore annexin V to the nuclei within 5 hours. Charcoal-treated serum cannot allow annexin V to return to the nucleus. Inhibition of protein synthesis with cycloheximide does not prevent the serum-induced return of annexin V to the nuclei. However, treatment of cells with genistein at a concentration specific for inhibition of tyrosine kinases (200 μM) inhibits the relocation of annexin V from cytoplasm to the nucleus. Thus, the cellular location of the annexin V depends on the growth state of the cells. It can be altered by the movement of this protein between the cytosol and the nucleus.
... (i) Both proteins have been shown to relocate in the same subcellular compartment, the nuclear membrane, after activation in many cell types (62)(63)(64)(65). This location may be relevant to their related biological function. ...
... In vivo, during cellular activation, calcium oscillations may reach 10 M [Ca 2ϩ ] i in specific subcellular domains located at the nuclear membrane and at the sarcoplasmic membrane (50). These locations of high [Ca 2ϩ ] i correspond with those of cPLA 2 and annexin V after cell stimulation (62)(63)(64)(65). ...
Annexin V belongs to a family of proteins that interact with phospholipids in a Ca²⁺-dependent manner. This protein has been demonstrated to have anti-phospholipase A2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA2 (cPLA2). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA2 activity after stimulation by calcium, GTPγS (guanosine 5′-O-(3-thiotriphosphate)), formyl-Met-Leu-Phe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca²⁺] of 3 and 10 μm. The mechanism of inhibition was also analyzed. cPLA2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca²⁺ site. We demonstrate that the Ca²⁺ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA2. Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA2 activity by depletion of the phospholipid substrate.
... In NRK cells annexin 6 was almost exclusively found associated with the prelysosomal compartment [7], This intracellular location does not rule out its presence at other sites such as the plasma membrane, and most probably reflects the various antibodies used, which may recognize different isoforms or conformations of annexin 6, or different cells analyzed. Besides, annexins I,II, IV, V, and VI relocate in response to rises in intracellular calcium; particularly, annexin 6 was shown to move from the perinuclear region to a more homogeneous distribution on the plasma membrane, in human fibroblasts [30,54], or to the sarcolemma to serve as a link of the cytoskeleton, in smooth muscle cells [20]. In addition, we now have evidence that annexin 6 may undergo ligand-induced translocation [42]. ...
... (1) the rise of intracellular Ca 2ϩ triggered by LDL [55][56][57] and (2) the reorganization of annexin 6 (relocate) according to Ca 2ϩ mobilization in the cell [20,54]. We conclude that, although annexin 6 might be dispensable (see work in knockout mice by Hawkins et al. [58]) for adult structure or development and/or even for those cellular aspects in which annexin 6 has been involved, in cells expressing annexin 6 it seems that it can be involved in intracellular trafficking from the endocytic compartment to the lysosomes. ...
Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx61–175), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 61–175. Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation.
... However, the properties of endogenous, cellular, annexin A5 are still largely unknown. Annexin A5 which was more often considered as a major intracellular Ca 2+ -binding protein [6] has been reported to be translocated from cytosol to plasma or nuclear membranes after a rise in intracellular 0008 Ca 2+ concentration [7][8][9]. It is known for inducing Ca 2+ influx by forming Ca 2+ channels [10] and for inhibiting enzyme activities linked to Ca 2+ activation such as phospholipase A2 and protein kinase C [11,12]. ...
... Taken together, these results strongly suggested that annexin A5 externalization was occurring very early during apoptosis process and was independent from that of PS. Dependent on an increase in [Ca 2+ ] i , translocation of annexin A5 to nuclear and/or cellular membrane has been reported in various cell types [7,8] and in cardiomyocytes during metabolic inhibition or heart failure [13,20,29]. Beyond translocation to membranes this study showed for the first time that annexin A5 was externalized in cardiomyocytes during apoptosis. ...
Annexins are Ca(2+)-dependent phospholipid binding proteins. Externalized annexin A5 has been recently suggested to have a proapoptotic effect. Our aim was to determine whether annexin A5, which is intracellular in cardiomyocytes, could be translocated and/or externalized and play a role during the apoptotic process.
Apoptosis was induced in rat cardiomyocytes by continuous incubation with staurosporine or 30 min treatment with H(2)O(2) and was measured by phosphatidylserine (PS) externalization, TUNEL staining and DNA ladder. Immunofluorescence labeling of annexin A5 was performed on permeabilized or nonpermeabilized cardiomyocytes.
Staurosporine or H(2)O(2) treatment of neonatal cardiomyocytes resulted in significant increases of apoptosis at 24 h, but H(2)O(2) treatment led to a faster and higher PS externalization than that observed with ST. In both neonatal and adult cardiomyocytes, annexin A5 was intracellular in control conditions but was found at the external face of sarcolemma during apoptosis. Furthermore, neonatal cardiomyocytes with externalized annexin A5 have apoptotic characteristics and their number increased with time. Interestingly, immediately after H(2)O(2) induction, the number of annexin A5-positive cells was higher than that of PS-positive cells (p</=0.05) and colabeling showed that half annexin A5-positive cells were PS-negative. We further demonstrated by immunoblots that free annexin A5 was absent from the media and could not be released from cardiomyocytes by washes at 1.8 mM Ca(2+). Removing annexin A5 by Ca(2+)-free washes 15 or 30 min after H(2)O(2) treatment or blocking externalized annexin A5 by antibodies lead to a significant decrease of apoptotic cardiomyocytes, cytochrome c release and caspase 3 activity.
This study indicated for the first time that annexin A5 was externalized at a very early stage of apoptosis and could have a proapoptotic effect in cardiomyocytes.
... The fluorescence spectroscopy method can quickly and qualitatively evaluate the wettability of formation 30 . Aromatic hydrocarbons and polar compounds in hydrocarbon spontaneously fluoresce when excited by ultraviolet light 31 . QGF-E spectrum represents the fluorescence characteristics of hydrocarbon adsorption on the surface of reservoir particles, which can be used to determine the present or residual oil reservoirs in exploration and drilling evaluation. ...
The study of natural gas accumulation process in tight formation has become the focus of the petroleum industry. One of the priorities is the effects of interactions in natural gas/water/rock system on hydrocarbon migration and accumulation process. On the macroscopic scale, we investigate the interactions in natural gas/water/rock system by formation fluorescence test and production data analysis. One the microscopic scale, the mechanisms are revealed by mathematical analysis and experimental methods considering the variation of geological temperature and pressure. The effects of interactions in natural gas/water/rock system are also simulated by numerical simulation. The results are visualized and quantified. A novel semi-analytical method based on a physical experiment is proposed to calculate the temperature- and pressure-dependent contact angle and interface tension which reflect the interactions in the natural gas–water–rock system. This semi-analytical is embedded in the numerical simulation during the simulation of the natural gas charging process. The results indicate that with the increase of geological temperature and pressure, the contact angle will increase and the interface tension between natural gas and water will decrease. The capillary resistance in the formation will be reduced. Since the decrease of capillary resistance, the natural gas can be charged into smaller pores, so that the actual charging threshold is lower than the one originally obtained under present reservoir conditions. After considering the temperature and pressure during the accumulation process, some sand bodies that were thought not to be charged may have natural gas accumulate.
... Ca 2+ -dependent membrane translocation is commonly observed in some ANX family proteins, including ANXA11 (20,21). To examine the impact of ALS-linked missense variants on Ca 2+ -dependent subcellular distribution of endogenous ANXA11, we performed immunocytochemistry using passage-matched control and patient fibroblasts carrying the p.G38R, p.H390P, or p.R456H variants. ...
Dysregulation of calcium ion homeostasis and abnormal protein aggregation have been proposed as major patho-genic hallmarks underpinning selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Recently, mutations in annexin A11 (ANXA11), a gene encoding a Ca 2+-dependent phospholipid-binding protein, have been identified in familial and sporadic ALS. However, the physiological and pathophysiological roles of ANXA11 remain unknown. Here, we report functions of ANXA11 related to intracellular Ca 2+ homeostasis and stress granule dynamics. We analyzed the exome sequences of 500 Korean patients with sALS and identified nine ANXA11 variants in 13 patients. The amino-terminal variants p.G38R and p.D40G within the low-complexity domain of ANXA11 enhanced aggregation propensity, whereas the carboxyl-terminal ANX domain variants p.H390P and p.R456H altered Ca 2+ responses. Furthermore, all four variants in ANXA11 underwent abnormal phase separation to form droplets with aggregates and led to the alteration of the biophysical properties of ANXA11. These functional defects caused by ALS-linked variants induced alterations in both intracellular Ca 2+ homeostasis and stress granule disassembly. We also revealed that p.G228Lfs*29 reduced ANXA11 expression and impaired Ca 2+ homeostasis, as caused by missense variants. Ca 2+-dependent interaction and coaggregation between ANXA11 and ALS-causative RNA-binding proteins, FUS and hnRNPA1, were observed in motor neuron cells and brain from a patient with ALS-FUS. The expression of ALS-linked ANXA11 variants in motor neuron cells caused cytoplasmic sequestration of endogenous FUS and triggered neuronal apoptosis. Together, our findings suggest that disease-associated ANXA11 mutations can contribute to ALS pathogenesis through toxic gain-of-function mechanisms involving abnormal protein aggregation.
... In muscle, annexins AS and A6 have been described in association with the membranes of the sarcoplasmic reticulum (SR) [7S], and in non-muscle cells annexin A6 has been associated with the endoplasmic reticulum (ER) [76]. Localisation to mitochondria and ER/SR could be related with a function in the regulation of the mobilisation of calcium from intracellular stores. ...
Annexin A11 is a ubiquitously expressed member of the annexin family of Ca2+-dependent phospholipid binding proteins, which lies at the root of the vertebrate annexin evolutionary tree. Annexin A11 is known to be localised to the nuclei of cells in culture, and to interact Ca2+-dependently in vitro with the S100 protein S100A6 and the penta EF-hand protein ALG-2 through its long glycine, proline and tyrosine-rich N-terminal domain, but very little is known about the physiological functions performed by this annexin in vivo. An investigation into the cellular roles of annexin A11 both in interphase and during cell division is presented in this thesis. Using various molecular and cell biology approaches, annexin A11 was identified in the nucleus and in cytoplasmic vesicles that appear to be transported along the microtubular network, linking annexin A11 function with intracellular membrane trafficking processes. Annexin A11 has also been identified in membrane domains involved in signal transduction at the basolateral membrane of A431 cells. A role for this annexin in membrane remodelling steps during cell growth is proposed in view of the accumulation of annexin A11-containing vesicles in the cytoplasm of non-contact inhibited cells and at the leading edge of monolayer outgrowths, together with the concentration of annexin A11 at the cell-cell contact sites in the plasma membrane of confluent monolayers. Annexin A11 relocates to the nuclear envelope and is tyrosine-phosphorylated in the presence of calcium in interphase cells, and invades the nuclear envelope at the invaginations created during nuclear envelope breakdown at the onset of mitosis, and is also detected at the nuclear envelope during nuclear envelope reassembly after cell division, making the nuclear envelope a specific target for annexin A11. The study of the function of annexin A11 during cell division led to the identification of an essential role for annexin A11 in the terminal phase of cytokinesis. Annexin 11 was observed to translocate from the nucleus to the spindle poles in metaphase, and then to the spindle midzone in anaphase, and to be recruited to the midbody in late telophase where it co-localises and associates with the mitotic kinesin- like protein CHO1. Depletion of annexin A11 by RNA interference has shown that the absence of this annexin leads to failure to establish a functional midbody, incomplete daughter cell separation, and ultimately cell death by apoptosis.
... Various Annexins have been shown to relocate during stress or Ca +2 upregulation. For example, Barwise & Walker (1996) demonstrated that Annexins II, IV and V relocated to plasma membrane when human foreskin fibroblasts were treated with calcium ionophore to increase the intracellular calcium levels. Chen et al. (2013) also suggested that plant annexins have a key role in the cross-talk between calcium regulation and ROS production under stress signaling. ...
The current study on putative rice annexin OsAnn5 was tried to know its functional role in the abiotic stress tolerance. For this an in silico analysis of its protein sequence and upstream region was carried out. This results in identification of several probable potential sites for post-translational modifications and cis-elements respectively. We have studied the effect of OsAnn5 in the amelioration of abiotic stress tolerance through heterologous expression in transgenic tobacco and E.coli. It is observed that OsAnn5 over expression leads to enhanced tolerance to abiotic stress through efficient scavenging of the ROS and balanced expression of SOD and CAT antioxidant enzymes in both the systems, under stress treatments. Fluorescent signal for transiently expressed EGFP:OsANN5 fusion protein was localized in the peripheral region of the onion epidermal cells under salt stress treatment. Expression analysis of OsAnn5 under ABA synthesis inhibitor, fluridone and salinity stress revealed that OsAnn5 appears to act through an ABA-independent pathway under salt stress and in support to this 35S:OsAnn5 transgenics seedlings exhibited less sensitivity to externally applied ABA.
... We show that endogenous AnxA5 is mainly cytosolic in myotubes, where it can represent up to 1% of the total protein mass. This huge intracellular amount of AnxA5 has also been reported in other cell types [28,34,46,59,60]. Endogenous AnxA5, as well as extracellular one, is specifically recruited to the wounded site in a few seconds after sarcolemma injury, in a Ca 2+ -dependent manner. ...
... The method for immunofluorescence microscopy was adapted from Barwise and Walker (Barwise and Walker, 1996) and Heggeness et al. (Heggeness et al., 1977). Cells were grown on glass coverslips in sixwell dishes overnight. ...
Cytosolic phospholipase A2-α (cPLA2-α) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA2-α is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA2-α is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA2-α. Here the nuclear localisation of cPLA2-α in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA2-α than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA2-α, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA2-α. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44MAPK and p38MAPK were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA2-α contains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA2-α. This, together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.
... Cells passaged 3-6 times were selected for the following experiments. The human foreskin fibroblasts (HSFs) were treated with 5.5 mM glucose, 30 mM glucose or 24.5 mM mannitol together with 5.5 mM glucose for three days [24,25]. ...
One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.
... Annexin V, a phospholipid-binding protein (Schlaepfer et al., 1992), is more concentrated in the nucleus than in the cytoplasm. If Ca 2 + concentrations in fibroblasts are increased, intranuclear annexin V relocates to the nuclear membrane, consistent with an important role in responding to the calcium signal within the nucleus (Barwise and Walker, 1996). Immunocytochemical studies found abundant annexin V expression by human lung, liver, kidney, skin, heart, uterus, spleen and skeletal muscle (Reutelingsperger et al., 1994) and a correlation of expression level with arrested cellular growth, suggesting a role in differentiation of glioma cells (Giambanco et al., 1993). ...
... The method for immunofluorescence microscopy was adapted from Barwise & Walker [53] and Heggeness et al. [54]. Cells were grown on glass coverslips in six-well dishes overnight. ...
Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.
... Studies have shown that the calcium-dependent translocation of AnxA6 to the plasma membrane occurs after an influx of extracellular calcium. [27][28][29] At the plasma membrane, AnxA6 functions as a membrane microdomain organizer and can regulate the activity of ion channels involved in both calcium entry and efflux in a cell-and context-specific manner. 30 -33 For example, calcium-induced binding of AnxA6 to the plasma membrane stabilizes the actin cytoskeleton and efficiently prevents store-operated calcium entry in HEK293 cells, whereas AnxA6 knockout mice show accelerated calcium efflux during cardiomyocyte contraction. ...
Vascular calcification is a pathological process common in patients with disorders of mineral metabolism and mediated by vascular smooth muscle cells (VSMCs). A key event in the initiation of VSMC calcification is the release of mineralization-competent matrix vesicles (MVs), small membrane-bound bodies with structural features enabling them to efficiently nucleate hydroxyapatite. These bodies are similar to MVs secreted by chondrocytes during bone development and their properties include the absence of calcification inhibitors, formation of nucleation sites, and accumulation of matrix metalloproteinases such as MMP-2. The mechanisms of MV biogenesis and loading remain poorly understood; however, emerging data have demonstrated that alterations in cytosolic calcium homeostasis can trigger multiple changes in MV composition that promote their mineralization.
... For example, annexin family proteins (two proteins in our study, Supplementary Table S1) are calcium-binding proteins that localize in the cytoplasm. However, when cytosolic Ca 2+ increases, they re-locate to the PM ( Barwise and Walker, 1996). We, of course, need to consider a slight contamination in the PM preparations from other organelles as evidenced by the presence of histones and ribosomal proteins. ...
The plasma membrane (PM) is the primary site of freezing injury in plants. To determine global changes in PM protein profiles in association with freezing tolerance development, proteome analysis of the purified PM of Arabidopsis suspension-cultured cells (T87 line) was conducted with label-free protein quantification technology. Freezing tolerance of Arabidopsis cells at the lag growth phase (8 d old) increased after cold acclimation (CA) or ABA treatment. Proteome analysis assigned 658 proteins in the PM in total, of which 45.3% (298 proteins) were predicted to have transmembrane domains. They were classified into several functional categories, with the primary categories being proteins in transporters, signal transduction, protein destination and storage, and cell structure. After CA, 271 proteins increased and 111 proteins decreased. ABA treatment resulted in 185 increased and 56 decreased proteins. Of these, 139 increased and 49 decreased proteins were identified in common after both CA and ABA treatment. In addition, there were proteins specifically expressed in cold- (132 increased and 62 decreased) or ABA- (46 increased and 7 decreased) treated cells. Collectively, our results clearly show that (i) responses of the PM proteome to CA and ABA treatment overlap substantially but, at the same time, some proteins exhibited different response patterns in each treatment; and (ii) the majority of ABA-responsive proteins are CA-responsive proteins but not vice versa, suggesting complex interactions of CA and ABA signaling pathways in the PM proteome responses.
... Our work supports the common hypothesis that, during mineralization processes of the cells, annexins translocate from the cytosol to special areas of the plasma membrane where MVs pinch off. The possible explanation is the relocation of annexins induced by elevating the concentration of intracellular calcium [32][33][34]. Therefore, we did not observe differences in annexins expression but differences of their content in MVs. ...
Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.
... In an effort to identify the membrane dynamics that result in Ca 2ϩ -dependent cell surface translocation of AnxA2, we used an ionophore-stimulated model in NIH 3T3 cells. Ionophoreinduced elevation of intracellular Ca 2ϩ has been a particularly valuable model to study Ca 2ϩ -dependent relocation of AnxA2 to cellular membranes (36). Relocation to the plasma membrane is related to several functions of AnxA2 including membrane trafficking, signaling events associated with Ca 2ϩ handling, and membrane-cytoskeletal interactions (37,38). ...
Annexin A2 (AnxA2), a Ca2+-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the
plasma membrane, AnxA2 associates in a Ca2+dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid
rafts is influenced not only by intracellular levels of Ca2+ but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along
the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular
endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular
environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca2+ ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like
characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These
results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes
of the endosomes to escape the endosomal degradation pathway. The Ca2+-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2.
... Previously, we found that only the NH 2 -terminal domain of ANXA6 was overexpressed in SCC (Lomnytska et al, 2010), which may indicate dysfunction of ANXA6 in SCC and explain the decrease in expression of full-length protein, as detected by IHC in cases of InvSCC. ANXA6 localises to the endoplasmic reticulum and plasma membrane (Barwise and Walker, 1996), and relocation of the protein from the cytosol to the plasma membrane is dependent on the elevation of Ca 2 þ influx (Buzhynskyy et al, 2009; Sztolsztener et al, 2009). We did not observe ANXA6 expression on plasma membranes of atypical cells, where expression was mainly cytoplasmic. ...
Cytology-based diagnostics of squamous cervical cancer (SCC) precursor lesions is subjective and can be improved by objective markers.
IHC-based analysis of ANXA6, HSP27, peroxiredoxin 2 (PRDX2), NCF2, and tropomyosin 4 (TPM4) during SCC carcinogenesis.
Expression of ANXA6, HSP27, PRDX2, and NCF2 in the cytoplasm of dysplastic cells increased from cervical intraepithelial neoplasia 2/3 (CIN2/3) to microinvasive cancer. Invasive SCC showed lower expression of TPM4 than CIN and normal epithelium. CIN2/3 with the highest sensitivity and specificity differed from normal epithelium by cytoplasmic expression of HSP27. Patients with cytoplasmic HSP27 expression in SCC deviating from that observed in normal epithelium had worse relapse-free (P=0.019) and overall (P=0.014) survival. Invasive SCC with the highest sensitivity and specificity differed from normal epithelium by expression of PRDX2 and TPM4 in the cytoplasm, from CIN2/3 by the expression of ANXA6 and TPM4 in the cytoplasm, and from microinvasive SCC by the expression of PRDX2 and ANXA6 in the cytoplasm. The number of sporadic ANXA6+ cells between the atypical cells increased from CIN2/3 to invasive SCC.
Detection of expression changes of the proteins ANXA6, HSP27, PRDX2, NCF2, and TPM4 in SCC precursor lesions may aid current cytological and pathological diagnostics and evaluation of prognosis.
An earlier investigation showed that annexin OsANN5 of rice was expressed to high levels in various stress treatments. In the present study, we have investigated the molecular mechanism underlying stress responses of OsANN5 through heterologous expression in transgenic tobacco and E. coli. E. coli cells expressing OsANN5 showed improved growth under high salt and heat stress treatments. Transgenic tobacco plants overexpressing OsANN5 exhibited tolerance to salinity, drought and osmotic stress during seed germination and post-germinative stages. The transgenic plants showed increased accumulation of proline, reduced chlorosis and membrane damage as compared to the wild type plants. Higher expression of OsANN5 also resulted in efficient scavenging of reactive oxygen species through the modulation of anti-oxidant enzymes, superoxide dismutase and catalase under stress treatments. OsANN5 exhibited calcium binding activity and under salt stress, relocated from the peripheral regions to cytosol. This suggests that OsANN5 has multiple functions in rice growth and stress tolerance. Promoter analysis of OsANN5 revealed the presence of several cis-acting elements that are well known to be involved in hormonal and stress response regulation. The expression patterns of OsANN5 in the presence of ABA synthesis inhibitor, fluoridone and salinity stress revealed that upregulation of OsANN5 transcripts is regulated by both ABA-dependent and independent pathways. This indicates OsANN5 may play a role in cross-talk between ABA-dependent and independent pathways during the abiotic stress response.
The annexins are a family of proteins that bind acidic phospholipids in the presence of Ca2+. The association of these proteins with the membranes of secretory granules and endosomes indicates these proteins may play a role in membrane trafficking. One member of the family, annexin II, can exist either as a monomer, heterodimer or heterotetramer in conjunction with the S100 protein p11. The ability of annexin II tetramer to bind both membranes and actin in a Ca2+-dependent manner has led to the hypothesis that annexin II may mediate between vesicle and/or plasma membranes and the cortical cytoskeleton. However, despite intensive biochemical characterisation in vitro, the function of this protein in vivo remains a mystery. In this study annexin II function in living cells was analysed in several different ways using green fluorescent protein (GFP) in full length annexin II-GFP chimeras and chimeras consisting of fragments of annexin II fused to GFP. Transfection of different cell lines with these annexin II-GFP constructs and fluorescence assisted cell sorting (FACS) allowed the generation of multiclonal cell populations expressing annexin II-GFP fusion proteins. These cell populations were analysed for effects on physiological functions - such as secretion (in the RBL cell line) or differentiation (of the PC12 cell line). This line of investigation did not yield evidence to support a role for annexin II in either of these processes. Using novel forms of microscopy the localisation of a full length annexin II- GFP chimera (NAII-GFP) was followed in single cells under physiological conditions. Under conditions of stress NAII-GFP was found to become incorporated into novel actin based structures, reminiscent of Listeria rockets, which propelled pinosomes through the cell interior. This form of vesicle locomotion is dependent on actin polymerisation and may represent a hitherto unrecognised form of vesicle transport.
Annexins are a family of calcium-binding proteins that bind membranes containing negatively charged phospholipids in a calcium-dependent manner. To date 12 human annexins have been identified, many of which are expressed in vascular endothelial cells. Annexins 1 and 5 have been suggested to play a role in the inhibition of endothelial cell cPLA₂, but this may be by substrate sequestration rather than a direct interaction between the proteins. Annexin 6 has been shown in vitro to produce a less thrombogenic surface when bound to phospholipids on the cell surface of endothelial cell membranes. The most interesting suggestion is that annexin 2 is a receptor for plasminogen and tissue plasminogen activator on the cell surface of human umbilical vascular endothelial cells (HUVECs), acting as a catalyst for the conversion of plasminogen to plasmin. When the vascular endothelium is damaged by oxidative stress this can lead to diseases such as atherosclerosis and hypertension. This damage is accompanied by changes in calcium responses and transcriptional regulation. This thesis reports an investigation into how the behaviour and expression of certain annexins is changed as a consequence of oxidative injury to vascular endothelial cells. HUVECs were cultured in 40% oxygen in a custom-built chamber to induce chronic oxidative stress over a period of 12 days. Immunostaining of annexins revealed altered patterns of translocation following stimulation with a range of different agonists, in cells cultured under normal and hyperoxic conditions. To investigate the underlying causes of these phenomena, changes in cellular levels of calcium, protein phosphorylation, MAP kinase activation and tyrosine phoshorylation were examined in oxidatively stressed cells. Levels of annexin mRNA and protein expression were also examined in HUVECs. The major findings of this work showed that annexin protein expression was unchanged, although prehaps with modest changes in mRNA expression for some annexins, in oxidatively stressed HUVECs. Changes in calcium mobilisation and other signalling events induced by oxidative stress were observed in conjunction with changes in annexin localisation. Annexin 5 showed the most dramatic change by translocating to the nuclei of oxidatively stressed cells, when it would not normally be seen in the nucleus. Studies in this thesis together with published work, indicate protein phosphorylation as being a major event leading to the changes observed in calcium signalling and annexin localisation in oxidative stressed HUVECs.
Members of the Annexin family of calcium-binding proteins are generally expressed in the cytosol of most eukaryotic cells from plants to humans. In vertebrates there are at least ten members of the family of which any one cell type expresses a selection. Annexins are characterised by the ability to bind calcium-dependently to negatively charged phospholipids, and by the presence of a 70 aa motif usually repeated four times. Numerous functions have been attributed to the annexins including calcium channel activity, regulation of exocytosis, phospholipase A2 inhibition and involvement in endocytosis. None of these postulated roles has been proved conclusively. This thesis describes work designed to define more rigorously the roles of members of this gene family in vivo by targeted disruption of the loci encoding the genes in the chick pre-B-cell DT40 lineage and mice. As a first step, targeting constructs were generated by a novel PCR-based method. Mice were then generated that have lost expression of Annexin VI, a unique member of the family in that it contains two instead of one set of four 70 aa repeats. One allele was disrupted in embryonic stem (ES) cells using the now well-documented method of homologous recombination, and genetically modified ES cells, identified by PCR, were then injected into blastocysts and reimplanted into recipient females. Chimaeric offspring that gave germline transmission were then bred to produce hetero- and finally homozygous null-mutant mice. Mice lacking Annexin VI are viable and fertile, and show no gross morphological defects. While no defects in B and T-cell development, or heart function were identified, adult male annexin VI knock-out mice gain weight at about half the rate of their wild-type counterparts, suggesting a novel role for annexin VI in animal physiology. In addition, clones of DT40 cells were generated with disruptions at both alleles of annexins II and V. The chick pre-B-cell DT40 lineage expresses several annexins including annexins I, II, V and VI. Targeted disruption is facilitated in this cell line by the expression of high levels of recombinase during IgG chain rearrangement. Loss of annexin II or V is also not lethal, but does lead to complex but distinct phenotypes involving calcium signalling, apoptosis and cell clumping. Detailed analysis of knock-out DT40 cells shows that these genes have non-redundant functions and play important roles in the cell. This work provides the experimental systems and information necessary to make significant progress in the understanding of the true roles of Annexins in cellular physiology.
The annexins or lipocortins are a multigene family of proteins that bind to acidic phospholipids and biological membranes in a Ca2+-dependent manner (Gerke and Moss 2002; Gerke et al. 2005; Raynal and Pollard 1994; Swairjo and Seaton 1994). Annexins are ubiquitous and characterized by an ability to bind to anionic phospholipids at membrane surfaces in response to elevated Ca2+. Annexins are amphipathic and distinct from soluble and integral membrane proteins, but share features of both (Kojima et al. 1994; Brisson et al. 1991). Annexins have molecular weights ranging between 30 and 40 kDa (only annexin 6 is 66 kDa) and possess striking structural features. The characteristic annexin structural motif is a 70-amino-acid repeat, called the annexin repeat. Four annexin repeats packed into an α-helical disk are contained within the C-terminal polypeptide core (Gerke and Moss 2002). While all annexins share this core region, aminoterminal domains of annexins are diverse in sequence and length (ranging from 11 to 196) on each annexin member. It is this diversity of N-terminal amino-acid sequence that gives the individual annexins their functional differences and biological activities and appears to differentiate the cellular function and location (Gerke and Moss 2002; Gerke et al. 2005; Raynal and Pollard 1994). Cysteine 198 is relatively conserved in annexins, and three of four cysteines (198, 242, and 315) in annexin A4 are conserved in annexin 3. Phospholipids are suggested to bind via hydrophilic head groups to annexins, and the phospholipid-binding region is proposed to be localized on the convex surface side where calcium-binding sites are located in the crystal structure of annexin 5 (Huber et al. 1990). The calcium- and phospholipid-binding sites are located in the carboxy terminal domains. Some of the annexins bind to glycosaminoglycans (GAGs) in a Ca2+- dependent manner. While annexin 2 has specific and high-affinity heparin-binding activity (Kassam et al. 1997), annexin A4 binds to heparin, heparan sulfate and chondroitin sulfate (CS) columns in a Ca2+- dependent manner, annexin 5 to heparin and heparan sulfate columns in a Ca2+-dependent manner and annexin 6 to heparin and heparan sulfate columns in a Ca2+-independent manner and to CS columns in a Ca2+-dependent manner (Ishitsuka et al. 1998) (see Table 21.1). Reports suggest that some annexin species may function as recognition elements for L-α-dipalmitoylphosphatidylethanolamine (PE)-derivatized GAGs under some conditions. The crystal structure of several of the annexins has been reported (Favier-Perron et al. 1996; Luecke et al. 1995; Swairjo et al. 1995). It has been established that the annexins are composed of two distinct sides. The convex side faces the biological membrane and contains the Ca2+- and phospholipid-binding sites. The concave side faces the cytosol and contains the N and C termini. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 bind also to carbohydrate structures suggesting that these annexin possess lectin-like domains.
Lipids include sterols, mono- and diglycerides, phospholipids, among others. They operate as structural components of cell membranes (Vol. 1 – Chap. 7. Plasma Membrane; Tables 2.1 and 2.2), contributors of energy metabolism, participants of intracellular transport (Vol. 1 – Chap. 9. Intracellular Transport), and signaling molecules.
Protein tyrosine kinases (PTK), i.e., enzymes that catalyze the phosphorylation of Tyr residues of proteins. are mainly associated with growth factor signaling. They actually modulate multiple cellular events, such as differentiation, growth, metabolism, and apoptosis. On the other hand, protein serine/ threonine kinases are principally related to second messengers, such as cyclic nucleotides cAMP (Sect. 11.1) and cGMP (Sect. 11.2), lipidic and related mediators diacylglycerol and inositol trisphosphate (Chap. 2), and calmodulin (Sect. 11.5.3).
Mitogen-activated protein kinase (MAPK) modules are commonly used to transduce signals aimed at regulating cell fate (cell differentiation, proliferation, senescence, and death) and inflammation. They mediate responses to extracellular signals, such as hormones, growth factors, and cytokines, as well as stresses and intercellular interactions.
Protein phosphorylation is a common, reversible, post-translational modification of proteins (Vols.1 – Chap. 5. Protein Synthesis and 3 – Chap. 1. Signal Transduction). Except pseudophosphatases, phosphatases removes a phosphate group from their substrates. They thus antagonize kinases that attach phosphate groups to Ser, Thr, and Tyr residues in the same substrates using ATP. Phosphorylated residues SerP, ThrP, and TyrP account for about 86, 12, and 2% of the phosphoproteome, respectively. Mammalian genomes encode about 100 protein Tyr kinases and phosphatases, and about 100 and about 25 protein Ser/Thr kinases and phosphatases.
Guanosine triphosphatases intervene in: (1) signal transduction from the intracellular edge of the plasma membrane and intracellular domain of transmembrane receptors; (2) protein synthesis at the ribosome (Vol. 1 – Chap. 5. Protein Synthesis); (3) control of cell division (Vol. 2 – Chap. 2. Cell Growth and Proliferation); (4) proper protein folding; (5) translocation of proteins through the membrane of the endoplasmic reticulum; and (6) vesicular transport within the cell (Vol. 1 – Chap. 9. Intracellular Transport).
Other important signaling mediators include: (1) endogenous, gaseous, diffusible messengers, or gasotransmitters, such as carbon monoxide (CO; Sect. 10.2), nitric oxide (NO; Sect. 10.3), and hydrogen sulfide (H2S; Sect. 10.4), in addition to oxygen and carbon dioxide, as well as several reactive oxygen (ROS) and nitrogen species (RNS; Sect. 10.6), that act as intra- and intercellular regulators; (2) membrane-bound enzymatic complexes such as the reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase or NOx; Sect. 10.5); (3) some coregulators, such as (protein kinase) A-kinase-anchoring proteins (AKAP; Sect. 10.8.4) and annexins (Sect. 10.8.5); and (4) transcription factors involved in stress response, such as the proteic complex nuclear factor κ light-chain-enhancer of activated B cells (NFκB; Sect. 10.9.1), the heterodimer hypoxia-inducible factors (HIF; Sect. 10.9.2), and members of the Forkhead box (Fox; Sect. 10.9.3) and P53 families (Sect. 10.9.4; Table 10.1).
Numerous protein serine/threonine kinases operate in signal transduction, either as initiating nodes, such as receptor (RSTK; Vol. 3 – Chap. 8. Receptor Kinases), or as intermediate nodes of signaling cascades, non-receptor (intracellular; NRSTK) protein Ser/Thr kinases. Typically, second messengers activate protein Ser/Thr kinases, whereas extracellular signals stimulate protein Tyr kinases (Chap. 4). For example, IκB kinases (IKK) of IκB inhibitors of nuclear factor-κB are serine kinases (Sect. 10.9.1).
Information on receptor ligand systems used by NK cells to specifically detect transformed cells has been accumulating rapidly. Killer cell lectin-like receptor subfamily K, member 1, also known as KLRK1, is the product of human gene. The KLRK1 has been designated as CD314 and contains a C-type lectin-like domain (CTLD). KLRK1 is also known as: KLR; NKG2D; NKG2-D; FLJ17759; FLJ75772; D12S2489E. Human NKG2D was originally identified in 1991 as an orphan receptor on NK cells (Houchins et al. 1991). Although genetically mapping near the C-type lectin receptors CD94 and NKG2A-E, the NKG2D activating NK cell receptor has little sequence homology with these receptors and is expressed as a homodimer that signals through DAP10 rather than CD94 (Chap. 30). NKG2D binds to two distinct families of ligands, the MHC class I chain-related peptides (MICA and MICB) and the UL-16 binding proteins (ULBP). These ligands are upregulated in cells that have undergone neoplastic transformation, and NK cytotoxicity on tumor cells correlates with tumor expression of MICA and ULBP. The NKG2D differs from other members of the NKG2 family in significant ways. They do not form heterodimers with CD94 on the cell surface. Instead, they are expressed as homodimers, and each homodimer associates noncovalently with a homodimer of the adaptor protein DAP-10. The cytoplasmic tail of DAP-10 carries a YxxM motif, which can recruit the regulatory subunit p85 of phosphatidylinositol-3 kinase and Grb2 (see also Chap. 30).
Multiple signaling processes are characterized by bursts of calcium ions that moves to specific locations for optimal activation of cell activity. Intracellular calcium regulates numerous protein functions in tiny cellular domains ( V1.2 channels form signaling clusters in plasmalemmal nanodomains. Fluorescence microscopy is aimed at imaging in real time communication within and between cells. Calcium fluxes through Ca\({}^{++}\) channels and gap junctions can be imaged once constitutive proteins (e.g., connexin) have been tagged with tetracysteines [1429].
Dual-specificity kinases (DSK; or Ser/Thr/Tyr kinases) phosphorylate their substrates on serine, threonine, and/or tyrosine residues. Dual-specificity kinases intervene in the regulation of cell growth, differentiation, and apoptosis. Dual-specificity kinases include, at least, members of the superfamily of mitogen-activated protein kinases as well as those of the family of glycogen synthase kinases.
Among kinases (or phosphotransferases) that transfer phosphate groups from donor molecules such as ATP or GTP to specific substrates include protein kinases that modify the activity of their proteic substrates. Kinases are used to transmit signals in cells. Other types of kinases act on amino acids, lipids, carbohydrates, and nucleotides, that are then used in signaling pathways or cell metabolism.
This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase α. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.
This chapter provides an up-to-date summary of recent developments in the field of annexin-nucleotide interactions and their
physiological significance. Since the first repons by Burgoyne and Geisow in the 1980s, a growing amount of evidence was published
suggesting that members of an evolutionary ancient family of calcium- and membrane-binding proteins may also bind nucleotides.
We propose that this property of annexins would serve as the molecular basis for their role as molecules connecting cellular
energy metabolism with various vital, nucleotide-dependent cellular processes, e.g., calcium homeostasis, vesicular trafficking
and signal transduction. The nucleotide-binding properties of annexins are evident from the results of experiments indicating
that some annexin isoforms bind in vitro ATP/GTP and/or cAMP (their radiolabelled, light-sensitive or fluorescent derivatives),
although within the annexin molecule there is no easily identified nucleotide-binding motif found in other families of nucleotide-binding
proteins characterized to date. In addition, annexins are involved in many energy-consuming processes and/or interact with
nucleotide-binding proteins and enzymes. In line with these observations are the results of intracellular localization of
annexins tightly associated with membranes of energy-transducing organelles, organelles involved in vesicular traffic and
membrane microdomains participating in signal transduction. The physiological significance of nucleotide-annexin interactions
remains, however, unclear. Perhaps observations describing human annexin VII as a Ca2+-dependent GIPase involved in membrane fusion, further extended to various plant annexin isoforms exhibiting myosin-like or
phospholipid-inhibited phosphodiesterase activity, provides a possible clue. As an alternative, for annexin V involved in
canillage calcification and the formation of voltage-dependent ion channels in membranes, the GTP regulatory mechanism of
ion channel activity was described. Recently, GTP-induced ion channel activity was also observed for human recombinant annexin
VI. It is also possible that the binding of nucleotides to annexins is responsible for the Ca2+-independent activities of these proteins.
Purpose: Cavernous nerve resection (CNR) in rats is a standard model of animal experiments on erectile dysfunction (ED) that occurs after radical prostatectomy (RP). Injured cavernous nerves after surgery can cause fibrosis and apoptosis that lead to penile structural changes that may be accompanied by alterations of protein expression. This study aimed to analyze the changes in protein after CNR in Wistar Kyoto rats. Materials and Methods: Using 8-week-old male Wistar Kyoto rats, sham and CNR operation under a microscope were performed. Two and 8 weeks after surgery, we applied 2-DE and MALDI-TOF/TOF (AB 4700) to identify differently expressed penile proteins after CNR. 2-DE gels were stained with silver nitrate and were analyzed with PDQuest. After in-gel digestion, peptide mass spectra were obtained by MALDI-TOF/TOF mass spectrometry in the positive ion reflector mode. The obtained data were screened with a rat database from both the NCBI and the Swiss-Prot/TrFMBL home page-Results: The proteins that were changed more than 1.5-fold compared with the sham group were annexin A4 and pyruvate kinase (PK). Annexin A4 was increased by 1.75-fold after 2 weeks, whereas PK was decreased by 4.16 after 8 weeks. These results were confirmed by immunohistochemistry. Conclusions: Annexin A4 in the CNR group was increased, which may be related to emiocytosis during apoptosis. The decrease in PK of the CNR group is assumed to be related to a decrease in efficacy during glycolysis. Further study will be needed to elucidate the molecular pathophysiology of ED after cavernous nerve injury.
The effect of protein kinase C(PKC) on the release of neurotransmitters from a number preparations, including sympathetic
nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between
effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The
enhancement of neurotransmitter release is discussed in relation to the effect of PKC on:
1.
Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of
large dense-cored vesicles to release sites on the plasma membrane.
2.
Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of
secretion.
3.
Ion channel activity, particularly Ca2+ channels.
Annexins are a family of 13 proteins known to bind phospholipids in a Ca2+-dependent way (1,2). They share a similar structure characterized by a conserved exterminai domain and a variable N-terminal domain. They are
thought to participate in a variety of membrane-related events such as exocytosis, endocytosis, binding to cytoskeletal proteins
but also to regulate protein activities. In the myocardium, annexin VI is the most abundant and has been localized in various
cell types including myocytes. It has been involved in the regulation of Ca2+ handling proteins and surexpression or knockout of the annexin VI gene leads to changes in Ca2+ homeostasis and contractility. Expression and localization of annexin VI is either faintly or not modified during heart failure.
Annexin V is mainly localized in the sarcolemma and T-tubules of cardiomyocytes and in endothelial cells. In the failing heart,
expression of annexin V is increased and localization becomes heterogenous leading to myocytes devoided of annexin V and interstitial
tissue strongly labelled. Annexin VII is known as a skeletal muscle annexin but is also expressed in the heart. It is the
third annexin which could be related to regulation of Ca2+ handling proteins because the knockout of the annexin VII gene shows a decrease in the force-frequency relationship in adult
cardiomyocytes. The major endothelial annexin is annexin II which is involved in membrane trafficking, collagen binding and
plasminogen activation. It is absent from myocytes. During heart failure, the increase in annexin II might play a role in
the extracellular matrix remodeling. From these studies, it is tempting to speculate that annexins, at least annexins V, VI
and VII, might play a role in the regulation of Ca2+ handling proteins during heart failure.
Key wordsCa2+ homeostasis and contractility–force-frequency relationship extracellular matrix remodeling
Annexin V relocates to specific cellular membranes on elevation of cytosolic calcium levels. There is good evidence for annexin
V binding to proreins involved in signal transduction including protein kinase C and cytosolic phospholipase A2, and to the cytoskeletal protein actin. In human plarelers annexin V relocation is in pan a phosphorylation-dependent process
that resulrs in binding of annexin V to the minor γ-isoform of actin.
The annexins, are a family of calcium ion (Ca2+)-binding proteins whose physiological functions are poorly understood. Although many diverse functions have been proposed
for these proteins, such as in vesicle trafficking, this review focuses on their proposed roles as Ca2+ or other ion channels, or as intracellular ion channel regulators. Such ideas are founded mainly on in vitro and structural
analyses, but there is increasing evidence that at least some members of this protein family may indeed play a part in intracellular
Ca2+ signaling by acting both as atypical ion channels and as modulators of ion channel activity. This review first introduces
the annexin family, then discusses intracellular localization, developmental regulation, and modes of membrane association
of annexins, which suggest roles in Ca2+ homeostasis. Finally, it examines the structural and electrophysiological data that argue for key roles for annexins in the
control of ion fluxes.
The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. 1998 Chapman & Hall
We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1%±0.78 of annexin V is present at the plasma membrane and 15.0%±0.82 in the region corresponding to the membrane cytoskeleton (10–80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7%±0.22 and 40.4%±0.53, respectively.
Serum starvation of MG-63 cells increases their doubling time from 24 h to 4 days. Cells grown in medium containing 10% fetal calf serum contain high levels of annexin V in the cell nucleus, whereas growth for 4 days in the absence of serum results in loss of nuclear annexin V from 72 ± 4% of cells. Many of the cells which still have nuclear annexin V under these conditions seem to have recently finished dividing. Refeeding cells with medium containing serum restores annexin V to nuclei within 5 h. Charcoal treatment removes factors from serum that are required to allow annexin V to return to the nucleus. Protein synthesis is not required for annexin V to return to nuclei since inhibition of protein synthesis with cycloheximide does not prevent the serum-induced return of annexin V to nuclei. This, and other evidence, indicates that the presence of annexin V in nuclei reflects translocation rather than catabolism and resynthesis. Inhibition of tyrosine kinase activities with genistein attenuates the relocation of annexin V from the cytoplasm to the nucleus. Thus, the nuclear location of annexin V is controlled by signaling pathways involving serum factors and tyrosine kinases. The results argue for an important role for annexin V in the cell nucleus.
We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.
Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs.
To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress.
Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites.
In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.
S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70(S6K1) isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85(S6K1) isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85(S6K1) substrates. Four novel putative p85(S6K1) substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.
Temporal changes in intracellular free Ca²⁺ concentration ([Ca²⁺]i) in cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca²⁺]i in each individual cell within a microscopic field. Responses to BK are heterogeneous with respect to the shapes of the [Ca²⁺]i time courses. Furthermore, the onsets of these responses follow a variable lag period such that the individual cell responses occur asynchronously. The asynchrony and heterogeneity of individual cell responses are not related to cell cycle differences since noncycling cells respond in a similar manner. When cells are ranked according to order of an initial response to BK (the first cell to respond is ranked 1, the second to respond is 2, etc.), restimulation of the same cells with BK elicits a similar order of cell responses, and the shape of the [[Ca²⁺]i time course of an individual cell is similar for both responses to BK. If cells that were stimulated with BK are washed and restimulated with thrombin (which produces [Ca²⁺]i changes similar to those induced by BK), the response order to thrombin does not correspond to the response order following BK stimulation. These data suggest that the asynchrony of [Ca²⁺]i changes induced by BK or thrombin is characteristic for each mitogen and may be determined by cell-to-cell variation in receptor number.
A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
The temporal and spatial dynamics of intracellular signals and protein effectors are being defined as a result of imaging using fluorescent reagents within living cells. We have described a new class of fluorescent analogues termed optical biosensors, which sense chemical or molecular events through their effects on protein transducers. One example of this new class of indicators is MeroCaM, an environmentally sensitive fluorophore which when it is attached to calmodulin reflects the activation of calmodulin by calcium in vitro. We report here that the rise in free calcium and MeroCaM activation occur in the same period during serum stimulation of quiescent fibroblasts. MeroCaM activation also correlates with the spatial pattern of increased free calcium and the contraction of transverse fibres during wound healing. Finally, migrating fibroblasts in the later stages of wound-healing exhibit an increasing gradient of free calcium and MeroCaM activation from the front to the rear.
A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.
Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.
Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.
We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.
Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. We have isolated and sequenced a set of overlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. These include endonexin II, lipocortin I, calpactin I heavy chain (p36), protein II, and calelectrin 67K. The Mr 51,000 synexin molecule can be divided into a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Analysis of the entire structure reveals possible insights into such diverse properties as voltage-sensitive calcium channel activity, ion selectivity, affinity for phospholipids, and membrane fusion.
Protein II isolated from porcine intestinal epithelium is a Ca2+-modulated lipid-binding protein. The amino acid sequence of porcine protein II reported here sheds new light on the properties of a multigene protein family which includes the tyrosine kinase substrates of the sarc gene (p36) and of the EGF-receptor (p35). The sequence consolidates the structural principle in which an amino-terminal tailpiece of variable length is followed by a core built from four internally homologous segments for those proteins in the 35-40 kd range. Sequence data also show that the core can now be described as two domains each containing one low and one high homology segment. This view accounts for two Ca2+ sites, lipid aggregation and F-actin bundling--when present--and suggests that properties of the cores in which protein II differs from p36 and p35 arise primarily from segments 1 and 2. The protease-sensitive tailpiece of protein II is very short and lacks the phosphorylatable tyrosine present in the larger tail domains of p36 and p35. It harbors, however, like the p36 domain, the major site for in vitro phosphorylation by the Ca2+- and lipid-activated protein kinase C. In protein II this site is most likely threonine 6. The sequence alignment also explains why protein II does not interact with a unique p11, a property probably specific for p36. Our results further suggest that liver endonexin may reflect two protein species both closely related to protein II.
Calpactins are a family of related Ca++-regulated cytoskeletal proteins. To analyze the expression and cytoskeletal association of calpactins we raised monoclonal antibodies with specificity for the heavy or light chains of calpactin I or to calpactin II. Comparison of the tissue distribution of calpactin I heavy and light chains by Western blots revealed that these subunits are coordinately expressed. Both soluble and cytoskeletal forms of the heavy chain of calpactin I were detected in human fibroblasts whereas only a soluble pool of calpactin II was found. These two forms of the calpactin I heavy chain differed both in their state of association with the light chain and in their rate of turnover. Both the soluble pool of the calpactin I heavy chain and calpactin II turned over three to four times faster than the cytoskeletal pool of heavy and light chains. Immunofluorescence microscopy revealed that the calpactin I light chain was present exclusively in the cytoskeleton whereas the calpactin I heavy chain distribution was more diffuse. No difference in the amount of light chain or the cytoskeletal attachment of phosphorylated calpactin I heavy chain was found in Rous sarcoma virus-transformed chick embryo fibroblasts compared with their normal counterpart. The antibody to the light chain of calpactin I was microinjected into cultured fibroblasts and kidney epithelial cells. In many cases antibody clustering was observed with the concomitant aggregation of the associated calpactin I heavy chain. The distribution of fodrin and calpactin II in injected cells remained unchanged. These results are consistent with the existence of two functionally distinct pools of calpactin I which differ in their association with the cytoskeleton.
Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.
The question of whether the Ca2+ ionophore A23187 affects collagen production relative to total protein synthesis or has possible effects on collagen degradation was investigated. Cultured normal human fibroblasts were incubated with radioactive proline, and the radioactivity of collagenase-sensitive and -resistant proteins was used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by A23187 in a dose-related manner, and 50% inhibition of basal collagen production was achieved with 0.6 microM A23187. There was a 70% decrease in the absolute rate of collagen production in the presence of 0.6 microM A23187 which represented a 4-fold greater inhibition of collagen production than of noncollagen protein production. The major mechanism for the decreased net production of collagen was decreased synthesis, rather than increased degradation. Ca2+ mobilization induced by cholecystokinin octapeptide was also associated with selective inhibition of collagen production in normal human fibroblasts. These studies establish that the Ca2+ ionophore A23187 induces a selective decrease in collagen polypeptide synthesis by normal human fibroblasts and suggest a modulatory role of Ca2+ on collagen metabolism.
Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.
The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Protein II isolated from porcine intestinal epithelium is a Ca2+‐modulated lipid‐binding protein. The amino acid sequence of porcine protein II reported here sheds new light on the properties of a multigene protein family which includes the tyrosine kinase substrates of the sarc gene (p36) and of the EGF‐receptor (p35). The sequence consolidates the structural principle in which an amino‐terminal tailpiece of variable length is followed by a core built from four internally homologous segments for those proteins in the 35‐40 kd range. Sequence data also show that the core can now be described as two domains each containing one low and one high homology segment. This view accounts for two Ca2+ sites, lipid aggregation and F‐actin bundling–when present–and suggests that properties of the cores in which protein II differs from p36 and p35 arise primarily from segments 1 and 2. The protease‐sensitive tailpiece of protein II is very short and lacks the phosphorylatable tyrosine present in the larger tail domains of p36 and p35. It harbors, however, like the p36 domain, the major site for in vitro phosphorylation by the Ca2+‐ and lipid‐activated protein kinase C. In protein II this site is most likely threonine 6. The sequence alignment also explains why protein II does not interact with a unique p11, a property probably specific for p36. Our results further suggest that liver endonexin may reflect two protein species both closely related to protein II.
The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.
We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.
Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild-type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.
The crystal structure of chicken annexin V has been solved by molecular replacement and refined at 2.25 angstrom. The final R factor is 19.7% with good geometry. The chicken annexin V structure is very similar to the human annexin V structure, with four similar domains each containing five helices. The structure includes three calcium ions in domains I, II, and IV, each bound by the characteristic K-G-X-G-T-(38 residues)-D/E motif. In view of the structural similarity between human and chicken annexin V, we suggest that they have a common vital function which developed early in evolutionary history.
33 kDa protein of neutrophil is a lipocortin-like protein, as proposed from its biochemical properties, amino acid composition, and the homology of its amino acid sequence to human lipocortin I. The localization and translocation of 33 kDa protein (p33) in blood cells of guinea pig were studied by immunoblotting (Western blotting) and immunocytochemical fluorescence methods using polyclonal and monoclonal mouse anti-p33 an-tibodies. The protein was determined to be present only in the cytoplasm of neutrophils, but not in cells such as the monocyte, lymphocyte, platelet, and other bone marrow cells. The translocation of the protein from cytoplasm to cell membrane was coupled with the increase in intracellular calcium ion and in superoxide generation induced by a chemotactic factor. These findings suggest that p33 may have an important role not only in the regulatory mechanism of phospholipase A2 (PLA2) activity but also in other transmembrane signaling.
Isolated plasma membranes attached to a solid substratum at 4 degrees C have numerous clathrin-coated pits. These pits initially are flat but become deeply invaginated after warming to 37 degrees C. The pits remain tethered to the membrane in this rounded condition unless supplied with ATP, Ca2+, and cytosol. We now show that when cytosol is treated to remove the Ca(2+)-dependent, phospholipid-binding protein annexin VI, coated pit budding no longer takes place. Addition of purified annexin VI back to the annexin VI-depleted cytosol restores budding activity to normal. Purified annexin VI alone shows only a modest budding activity, suggesting that the cytosol contains a factor(s) in addition to annexin VI that is required for full activity. Cytosol-dependent activation of annexin VI requires both ATP and Ca2+. Annexin VI appears to be not only an active component in the detachment of coated pits from the membrane but also a site for regulating the formation of coated vesicles.
Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.
The annexins are a group of homologous proteins that bind phospholipids in the presence of calcium. They may provide a major
pathway for communication between cellular membranes and their cytoplasmic environment. Annexins have a characteristic "bivalent"
activity in the sense that they can draw two membranes together when activated by calcium. This has led to the hypothesis
that certain members of this protein family may initiate contact and fusion between a secretory vesicle membrane and the plasma
membrane during the process of exocytosis.
The subcellular localization of annexin V in cultured human umbilical vein endothelial cells, epithelial cells and fibroblasts was examined. Indirect immunofluorescence and immunoblotting studies using affinity-purified anti-annexin V antibodies revealed that annexin V is located within the cytoplasm and nucleus of these cells. Further examination and direct binding studies showed that annexin V within the nucleus is associated with the nucleolus. These findings suggest that annexin V may play a role in a nucleolar function, such as ribosome assembly and transport.
Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.
After phagocytosis, killing and digestion of ingested microorganisms depend on fusion of phagocytic vesicle membranes with membranes of intracellular vesicles (azurophil and specific granules). There is considerable evidence that phagosome-granule membrane fusion is regulated by transient increases in intracellular ionized Ca2+. In previous studies, we found that a cytosolic Ca2(+)-dependent membrane-binding protein, annexin III, represents over 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro. To determine whether annexin III localizes to the region of phagosome-granule membrane fusion in vivo, we used a monospecific polyclonal antibody to stain fixed, permeabilized neutrophils that had ingested opsonized yeast. We found that annexin III concentrates in the region surrounding the phagosome. Annexin III was concentrated ninefold in the periphagosomal region compared with the cell body, as demonstrated by laser scanning confocal microscopy. Periphagosomal translocation of annexin III occurred whether yeast were opsonized with IgG, complement, or both, and persisted for at least 1 h after phagocytosis. This is not a general phenomenon, inasmuch as calmodulin was as abundant in the cell body as in the periphagosomal region. These findings imply that annexin III plays a specialized role in the metabolic and structural events that accompany phagocytosis.
Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.
Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specific calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.
Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.
Stimulation of bovine adrenal chromaffin cells results in a rise in the concentration of cytosolic calcium which triggers the release of catecholamines by exocytosis. Several cytosolic proteins that bind to secretory granule membranes in a calcium-dependent manner have been implicated in exocytosis and some belong to a family of calcium-binding proteins, the annexins. One of these, calpactin, is a tetramer consisting of two heavy and two light chains (relative molecular masses 36,000 and 10,000 respectively) and can aggregate and fuse membranes in vitro in the presence of arachidonic acid. Calpactin is found at the cell periphery and is phosphorylated when chromaffin cells are stimulated. We show here that both calpactin and calpactin heavy chain (p36) reconstitute secretion in permeabilized chromaffin cells in which secretion has been reduced as a result of leakage of cellular components. This effect is inhibited by an affinity-purified antibody against p36. Secretion from permeabilized cells is inhibited by a synthetic annexin-consensus peptide, but not by a nonspecific hydrophobic peptide; this inhibition is reversed by p36. Our results indicate that either calpactin or p36 is essential for exocytosis.
p36, a member of the family of Ca2+/lipid-binding proteins, is a major cellular substrate for the tyrosine kinase encoded by the src oncogene. It occurs in two distinct physical states, as either a monomer or a heterotetramer (protein I), which comprises two copies each of p36 and a p11 polypeptide. Immunofluorescence microscopy and cell fractionation studies suggest that p36 and p11 are located underneath the plasma membrane. To investigate whether p36 is indeed associated with the plasma membrane, we have examined its cellular distribution at the electron microscopic level with gold-labeled antibodies. In human fibroblasts, p36 is clearly associated with the cytoplasmic side of the plasma membrane and shows a uniform and regular distribution. Decoration with monoclonal antibodies against p11 reveals the same distribution, suggesting that the p36(2)p11(2) complex (protein I) occurs in the cell in a strict association with the plasma membrane. Titration experiments show that this association is Ca2+ dependent and still occurs at physiological Ca2+ concentrations (10(-7) M). Fodrin, a non-erythroid spectrin, known to bind p36 in vitro, shows a very similar distribution on the cytoplasmic side of the plasma membrane. The results suggest that in a resting and unstimulated cell p36 and p11 reside as a complex bound to the inner side of the plasma membrane.
Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).
Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.
Human placental lipocortin is a high-affinity substrate for rat brain protein kinase C in vitro with phosphorylation occurring on serine and threonine residues in a ratio of approximately 2 to 1. Comparison of the ability of various N-terminal-truncated derivatives of lipocortin to serve as phosphorylation substrates, and direct analysis of the N-terminal peptides cleaved from 32P-labeled lipocortin, indicated that threonine-24, serine-27, and serine-28 were the phosphorylation sites. The possibility is discussed that a lysine residue near the carboxy side of the phosphorylation site was involved in lipocortin interaction with the catalytic site of protein kinase C.
p36, a major cytoplasmic substrate of pp60 src kinase, is present beneath the plasma membrane. It can be isolated either as a monomer or as a heterotetramer (protein I) containing two copies each of p36 and a unique p11 polypeptide. To compare the expression rules of p36 and p11 as well as their cellular distributions, monoclonal antibodies to the two porcine proteins were isolated. In tissue culture cells p11-specific antibodies decorated the same submembranous compartment previously seen with antibodies to p36 and fodrin or spectrin and followed the p36 images under all fixation/extraction conditions tested. Immunofluorescence microscopy on tissue sections showed coincident expression patterns of both proteins confirming and extending previous results with p36 antibodies. Antibodies with limited cross-species reaction have been used to trace the fate of porcine p11 and p36 injected into cultured cells. Both proteins are incorporated in the submembranous compartment, where they remain in Triton cytoskeletons prepared in the presence but not in the absence of Ca2+. The incorporation of p36 in vivo conforms with its Ca2+-dependent binding to actin, fodrin, and certain phospholipids in vitro. In contrast, the incorporation of p11 seems to depend on an in situ interaction with p36 or an exchange with endogenous p11 present on p36. The combined results indicate a strong coupling of p11 and p36 in cellular compartmentalization and tissue differentiation.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
This review suggests that the intracellular functions of calcium are best understood in terms of calcium's functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.
The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.
The avian sarcoma virus-transforming gene product (pp60src) appears potentially able to mediate cell transformation via phosphorylation since it is tightly associated with a protein kinase activity. We have searched for and have been able to identify a normal cellular protein that appears to be a substrate of pp60src. The phosphorylation of this protein (34K) in transformation-specific in ASV-transformed cells of both avian and mammalian origin. Moreover, the 34K polypeptide serves as a substrate for the pp60src phosphotransferase activity in vitro and is phosphorylated at a site identical to the major site of phosphorylation in vivo. These data suggest that upon transformation the 34,000-dalton protein is phosphorylated directly as a result of pp60src activity.
Cell transformation by ASVs is the consequence of the expression of a single viral gene, termed src, for sarcoma induction (Vogt 1977; Hanafusa 1977). The product of the src gene is a 60,000-dalton phosphoprotein termed pp60src (Purchio et al. 1978). Extensively purified preparations of pp60src exhibit phosphotransferase activity for various protein substrates (Erikson et al. 1979 a, b; Collett et al. 1980), implying that phosphorylation of cellular proteins may play a role in transformation. Evidence that pp60src does mediate transformation via phosphorylation, however, requires the identification of a cellular protein(s) that is (are) phosphorylated directly as the result of pp60src activity both in cells and in vitro and the correlation of this phosphorylation with a phenotype of transformation.
The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.
Membrane vesicles derived from the apical side of procine intestinal epithelial cells retain, after demembranation in the presence of calcium, two major proteins (I, II) which are released by the addition of calcium chelators. We have purified and characterized these two calcium-binding proteins. Protein I has a mol. wt. of 85 000 and contains two copies of a 36-K subunit and an additional 10-K subunit. It binds in a calcium-dependent manner to F-actin as well as to non-erythroid spectrin. Immunofluorescence microscopy reveals protein I-related antigens in the terminal web of the intestinal cell and in a submembraneous cortical layer in various tissue culture cells. Biochemical and immunological results document that the 36-K subunit of protein I is identical with the cellular p36K recognized as a major substrate for tyrosine phosphorylation by the sarc gene kinase in Rous sarcoma virus-transformed cells. The biochemical properties of protein I agree with its location seen in immunofluorescence microscopy and cell fractionation and suggest that the actin-spectrin network in the cortical layer may be affected by virus transformation.