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PubMed2001

Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

3T3 Cells↗
PubMed2002

Linkage of caspase-mediated degradation of paxillin to apoptosis in Ba/F3 murine pro-B lymphocytes.

We have cloned the complete cDNA from mouse paxillin, a 68-kDa adapter protein found in focal adhesions. We found that paxillin was degraded by caspases in Ba/F3 cell apoptosis induced by withdrawal of interleukin-3 (IL-3), a survival factor for this cell, and by ionizing radiation. Also, paxillin was degraded in vitro by incubation with recombinant caspase-3. Western blot analyses of degradation products of overexpressed green fluorescence protein-tagged paxillin and site-specific mutants demonstrated that Asp-102 and Asp-301 were early caspase cleavage sites, and Asp-5, Asp-146, Asp-165, and Asp-222 were late cleavage sites. Overexpression of paxillin delayed apoptosis of Ba/F3 after IL-3 withdrawal. Furthermore, this anti-apoptotic effect of paxillin was augmented by a triple mutation in aspartic acids at caspase cleavage sites. These results suggest that paxillin plays a critical role in cell survival signaling and that the cleavage of paxillin by caspases might be an important event for focal adhesion disassembly during cell apoptosis, contributing to detachment, rounding, and death.

Animals↗
PubMed1991

Paxillin is a major phosphotyrosine-containing protein during embryonic development.

Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development.

Animals↗
PubMed2000

Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M. C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851-863). In this study, we report the cloning and initial characterization of a new paxillin LD motif-binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell-cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

Actinin↗
PubMed2001

Paxillin: application of immunohistochemistry to the diagnosis of chromophobe renal cell carcinoma and oncocytoma.

Paxillin is a cytoskeletal protein that was recently identified as a component of focal adhesions and links between F-actin and integrin. In this study, 91 renal tumors--65 conventional renal cell carcinomas (RCCs), 14 papillary RCCs, 6 chromophobe RCCs, 4 collecting duct carcinomas, 2 oncocytomas--were investigated for the immunohistochemical expression of paxillin. In a normal kidney, paxillin was predominantly expressed in the cytoplasm of distal tubules, loops of Henle, collecting ducts, and vascular smooth muscle cells. In all of the chromophobe RCCs and oncocytomas, strong expression of paxillin was observed in the tumor cytoplasm. In contrast to these tumors, conventional RCCs, papillary RCCs, and collecting duct carcinomas showed negative reactions for paxillin except for one case in each subgroup with weak reactivity. An immunoblot analysis confirmed the presence of paxillin in healthy kidney, chromophobe RCC, and oncocytoma. These data suggest that paxillin possibly plays a role in signal transductions as a focal adhesion intervening between tumor cells and the extracellular matrix in renal tumors with collecting duct phenotypes such as chromophobe RCCs and oncocytomas, but not in conventional RCCs. In addition, paxillin may be an available marker in distinguishing chromophobe RCCs from conventional or papillary RCCs.

Adenoma, Oxyphilic↗
PubMed1995

pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.

Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

Amino Acid Sequence↗
PubMed2001

Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase.

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

3T3 Cells↗
PubMed2001

Protein tyrosine phosphatase phi regulates paxillin tyrosine phosphorylation and mediates colony-stimulating factor 1-induced morphological changes in macrophages.

Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase phi (PTP phi). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP phi and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTP phi induces cell rounding and ruffle formation, while C325S PTP phi has no effect. In contrast, in CSF-1-starved cells, C325S PTP phi behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP phi increases adhesion in cycling cells, while WT PTP phi enhances motility. In WT PTP phi-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTP phi-expressing cells. Moreover, a complex containing PTP phi, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTP phi selectively binds paxillin and Pyk2 in vitro. Although PTP phi and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTP phi in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.

Blotting, Western↗
PubMed2005

Interaction of paxillin with poly(A)-binding protein 1 and its role in focal adhesion turnover and cell migration.

We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on two-dimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration.

Active Transport, Cell Nucleus↗
PubMed1995

Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells.

Angiotensin II is a potent vasoconstrictor that has been also implicated in vascular hyperproliferative diseases, including atherosclerosis and restenosis following angioplasty. Treatment of cultured, serum-starved rat aortic smooth muscle cells with angiotensin II causes rapid protein tyrosine phosphorylation that precedes cell mitogenesis. We have identified two of the phosphoproteins as paxillin (75 kilodaltons) and the tyrosine kinase pp125Fak, both components of actin-associated focal adhesion sites. Angiotensin II stimulated a 5-fold increase in the tyrosine phosphorylation of paxillin and a smaller (1.5-fold) increase in pp125Fak tyrosine phosphorylation. Paxillin tyrosine phosphorylation was evident within 1 minute, and was maximal after 10 minutes. Similar elevated protein tyrosine phosphorylation levels of paxillin were obtained with exposure of the rat aortic smooth muscle cells to peptides endothelin-1 and alpha-thrombin that function, as angiotensin II, through binding to members of the seven transmembrane domain G protein coupled receptors. Angiotensin II treatment also stimulated the production of a well-ordered actin-containing stress fiber network and prominent paxillin-containing focal adhesions. The focal adhesions stained intensely with anti-phosphotyrosine antibody suggesting the tyrosine phosphorylation of paxillin and cytoskeletal reorganization were tightly coupled. Angiotensin II receptor occupancy has been shown previously to lead to protein kinase C activation. However, compared to angiotensin II stimulation, a smaller, delayed increase in paxillin tyrosine phosphorylation was observed following direct protein kinase C activation by the phorbol ester phorbol 12-myristate-13-acetate. Paxillin tyrosine phosphorylation was selective for certain agonists since no increase in tyrosine phosphorylation of this protein was observed following exposure to the potent mitogen PDGF. Thus, actin-based cytoskeletal changes involving sites of cell adhesion to the extracellular matrix may play an important role in normal and pathophysiologic smooth muscle cell growth regulation in response to certain angiotensin II-type vasoactive agonists.

Angiotensin II↗
PubMed1999

Transcriptional up-regulation of paxillin expression by heregulin in human breast cancer cells.

Activation of heregulin (HRG) signaling has been implicated in the development of aggressive phenotype in breast cancer cells. The mechanisms through which HRG regulates the progression of breast cancer cells to a more invasive or motile phenotype are currently unknown. Because the process of cell migration must involve dynamic changes in the formation of new focal adhesions at the leading edge and dissolution of preexisting focal points, we explored the potential HRG regulation of paxillin, a major component of focal adhesion. Here, we report that HRG stimulation of noninvasive breast cancer MCF-7 cells resulted in the up-regulation of paxillin mRNA and protein. The observed HRG stimulation of paxillin mRNA expression was completely blocked by actinomycin D (a transcriptional inhibitor) as well as by cycloheximide (a protein synthesis inhibitor), suggesting the involvement of an inducible protein factor(s) and transcriptional regulation of paxillin mRNA by HRG. Extension of these observations to other HRG-responsive human cell lines also demonstrated that HRG has a significant capacity to up-regulate the paxillin expression. Furthermore, the levels of paxillin expression were closely linked with the coexpression of human epidermal growth factor receptor 2 (HER2)/HER3 receptors in breast cancer cell lines and in grade III human breast tumors. This study is the first demonstration of regulation of paxillin expression by a polypeptide growth factor, and it suggests a potential role for paxillin in the HER2 pathway in breast cancer.

Breast Neoplasms↗
PubMed2000

Association of beta 1 integrin with focal adhesion kinase and paxillin in differentiating Schwann cells.

Schwann cells (SCs) differentiate into a myelinating cell when simultaneously adhering to an axon destined for myelination and basal lamina. We are interested in defining the signaling pathway activated by basal lamina. Using SC/sensory neuron (N) cocultures, we identified beta1 integrin and F-actin as components of a pathway leading to myelin gene expression and myelination (Fernandez-Valle et al., 1994, 1997). Here, we show that focal adhesion kinase (FAK) and paxillin are constitutively expressed by SCs contacting axons in the absence of basal lamina. Tyrosine phosphorylation of FAK and paxillin increases as SCs form basal lamina and differentiate. FAK and paxillin specifically coimmunoprecipitate with beta1 integrin in differentiating SC/N cocultures but not SC-only cultures. Paxillin coimmunoprecipitates with FAK and fyn kinase in differentiating SC/N cocultures. A subset of tyrosine-phosphorylated beta1 integrin, FAK, and paxillin molecules reside in the insoluble, F-actin-rich fraction of differentiating cocultures. Cytochalasin D, an actin depolymerizing agent, decreases tyrosine phosphorylation of FAK and paxillin and their association with beta1 integrin and causes a dose-dependent increase in the abundance of insoluble FAK and paxillin complexes. Collectively, our work indicates that beta1 integrin, FAK, paxillin, and fyn kinase form an actin-associated complex in SCs adhering to basal lamina in the presence of axons. This complex may be important for initiating the process of SC differentiation into a myelinating cell.

Animals↗
PubMed1994

The aggregation of the high affinity IgE receptor induces tyrosine phosphorylation of paxillin, a focal adhesion protein.

Tyrosine phosphorylation of proteins is an essential component of high affinity IgE receptor (Fc epsilon RI) signaling and secretion. This signaling and secretion is also dependent on the organization of the cytoskeleton. Here we report that the aggregation of Fc epsilon RI on rat basophilic leukemia cells results in tyrosine phosphorylation of the cytoskeletal protein, paxillin. Tyrosine phosphorylation of paxillin is a relatively late event after Fc epsilon RI aggregation. Both the direct increase in intracellular Ca2+ with calcium ionophore and the activation of protein kinase C (PKC) with PMA induced tyrosine phosphorylation of paxillin. The optimal tyrosine phosphorylation of paxillin by Fc epsilon RI aggregation required PKC and extracellular Ca2+. However, there was also Fc epsilon RI-mediated tyrosine phosphorylation of paxillin independent of Ca2+ influx or PKC activation. By fluorescent microscopy, cell stimulation induced a redistribution of paxillin toward the periphery of the cells. Although Fc epsilon RI aggregation induced tyrosine phosphorylation of paxillin in nonadherent cells, adherence markedly enhanced this phosphorylation. Together, the data suggest a role for paxillin in Fc epsilon RI signaling.

Animals↗
PubMed2000

Differential tyrosine phosphorylation of paxillin in human corneal epithelial cells on extracellular matrix proteins

Purpose: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion.Methods: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and Western blotting to determine tyrosine phosphorylation of paxillin.Results: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix. Conclusion: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.

Journal Article↗
PubMed1999

Characterization of paxillin LIM domain-associated serine threonine kinases: activation by angiotensin II in vascular smooth muscle cells.

Recently we reported a novel means of regulating LIM domain protein function. Paxillin LIM zinc-finger phosphorylation in response to cell adhesion regulates the subcellular localization of this cytoskeletal adaptor protein to focal adhesions, and also modulates cell adhesion to fibronectin (Brown et al. [1998] Mol. Biol. Cell 9:1803-1816). In the present study, we characterize further the protein kinases that phosphorylate paxillin LIM2 on threonine and LIM3 on serine. Analysis of the subcellular distribution of the LIM kinases demonstrated that the LIM3 protein kinase, but not the LIM2 kinase, resides within a detergent-insoluble fraction. The activities of the paxillin LIM domain kinases are differentially regulated during embryogenesis, and analysis of tissue distribution indicated a specificity in expression patterns between the LIM2 and LIM3 kinases. In addition, these protein kinases were refractory to inhibition by a panel of broad-spectrum serine/threonine kinase inhibitors, suggesting a novel derivation. The paxillin protein kinase activities were stimulated in serum-starved CHO.K1 cells by the mitogen phorbol myristate acetate (PMA), and by PMA and angiotensin II in rat aortic smooth muscle cells. In vivo labeling, phosphoamino acid analysis, and phosphopeptide mapping of paxillin immunoprecipitated from angiotensin II-stimulated smooth muscle cells confirmed an induction of paxillin serine/threonine phosphorylation and supports the contention that these newly identified paxillin kinases are dynamic components of growth factor signaling through the cytoskeleton.

Angiotensin II↗
PubMed2000

Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini.

Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in [Ca(2+)](i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.

ADP Ribose Transferases↗
PubMed1999

Enhanced expression of the cytoskeleton-associated proteins paxillin and focal adhesion kinase in glomerular immune injury.

In a rat model of glomerular immune injury induced by antibody against the glomerular basement membrane (GBM), we assessed changes in the levels and in the extent of tyrosine phosphorylation of two cytoskeleton-associated proteins, focal adhesion kinase (FAK) and paxillin. Glomeruli were isolated 2, 7, and 14 days after the administration of a rabbit anti-rat GBM antibody that induced proliferative nephritis and proteinuria. FAK and paxillin levels in glomerular protein lysates were assessed by immunoprecipitation followed by Western blot analysis. Changes in the tyrosine phosphorylation of immunoprecipitated paxillin and FAK were assessed by Western blot analysis with antiphosphotyrosine antibodies. Glomerular levels of FAK and paxillin were increased in nephritic glomeruli as compared with non-nephritic controls at all time points. There was a discordant increase in the tyrosine phosphorylation levels of paxillin and FAK; the increase in the tyrosine phosphorylation of FAK was sustained and peaked on day 7 of immune injury, whereas that of paxillin was short-lived and peaked on day 2 of injury. We propose that these changes in FAK and paxillin expression and tyrosine phosphorylation reflect interactions between glomerular cells and accumulating extracellular matrix proteins in the course of immune injury, or they constitute parts of wider signaling events within the nephritic glomerulus that involve the cytoskeleton.

Animals↗
PubMed1998

Are tyrosine phosphorylation of p125(FAK) and paxillin or the small GTP binding protein, rho, needed for CCK-stimulated pancreatic amylase secretion?

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin.

ADP Ribose Transferases↗