Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.     

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.     

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.     

Obligatory role for phospholipase C-gamma(1) in villin-induced epithelial cell migration

While there is circumstantial evidence to suggest a requirement for phospholipase C-₁ (PLC-₁) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-₁ play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-₁. Inhibition of villin phosphorylation prevented villin-PLC-₁ complex formation as well as villin-induced cell migration. The absolute requirement for PLC-₁ in villin-induced cell migration was demonstrated by measuring cell motility in PLC-₁^–/– cells and by downregulation of endogenous PLC-₁. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-₁ at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration. fluorescence resonance energy transfer; actin     

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-gamma 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-gamma 1 and villin, both of which participate in the initiation of protrusion.     

Identification of a functional switch for actin severing by cytoskeletal proteins

Actin severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microM). Here we demonstrate that high calcium releases an autoinhibited conformation in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever actin in vivo.     

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.     

Janus kinase 3 regulates interleukin 2-induced mucosal wound repair through tyrosine phosphorylation of villin

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cell-specific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin.     

PLC-gamma1 signaling pathway and villin activation are involved in actin cytoskeleton reorganization induced by Na+/Pi cotransport up-regulation

BACKGROUND: The brief incubation of opossum kidney (OK) cells with low P(i) results in Na+/P(i) cotransport up-regulation and in substantial, but transient, cytoskeletal reorganization. In this study, we examined signaling events involved in the depolymerization of microfilaments. RESULTS: Confocal laser scanning microscopy, immunoblot and immunoprecipitation experiments revealed villin co-localization with mainly actin short filaments and monomers, indicating that under the conditions used, villin acted as an actin-severing protein. Further analysis revealed that low concentrations of extracellular phosphate resulted in phospholipase Cgammal (PLC-gammal) translocation to the actin cytoskeleton, without increases in its tyrosine phosphorylation. Additionally, tyrosine phosphorylation of a portion of insoluble villin was increased; whereas, only tyrosine phosphorylated villin associated with PLC-gammal. Although, tyrosine phosphorylation of PLC-gammal was not observed during Na+/P(i) cotransport up-regulation, genistein treatment abolished the enzyme's translocation to the actin cytoskeleton, as well as its association with villin. In addition, villin was found to associate with the 85-KDa subunit (p85) of phosphatidylinositol (PI)-3 kinase, concomitant with PLC-gammal, in the cytoskeletal fraction of Na+/P(i) cotransport up-regulated cells. CONCLUSIONS: Our observations suggest a signaling mechanism linking low ambient P(i) levels to the acute up-regulation of its cotransport with sodium and the depolymerization of the subcortical actin cytoskeleton.     

Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton

Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP(2) has never been characterized. In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin. Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics. Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin. Synthetic peptides analogous with these sites confirmed the binding domains. Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2). By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin. In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin. Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin. Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).     

Novel association of the src family kinases, hck and c-fgr, with CCR3 receptor stimulation: A possible mechanism for eotaxin-induced human eosinophil chemotaxis.

The chemokine eotaxin is a potent and relatively eosinophil-specific chemoattractant implicated in the cell migration to inflammatory sites in allergic diseases. Eotaxin exerts its activity solely through the CCR3 receptor, but the signaling pathways are poorly defined. In this study, we show that eotaxin induces an increase in tyrosine phosphorylation of multiple cellular proteins in normal human eosinophils. Eotaxin-dependent tyrosine phosphorylation was detected 1 min after stimulation and increased for at least 15 min with kinetics similar to those of eotaxin-induced cell shape changes. Herbimycin A, a tyrosine kinase inhibitor, blocked both eotaxin-induced tyrosine phosphorylation and cell shape changes as well as chemotaxis. Immunofluorescence microscopy analyses showed that eotaxin-induced cell shape changes were accompanied by redistribution of tyrosine-phosphorylated proteins and F-actin reorganization that were sensitive to herbimycin A. Coimmunoprecipitation studies revealed that binding of eotaxin to CCR3 greatly enhanced association of the Src family kinases, Hck and c-Fgr, with CCR3 after internalization of CCR3. These results may indicate that recruitment of Hck and c-Fgr to CCR3 in a compartment triggers tyrosine phosphorylation, leading to rapid cell shape changes required for cell migration.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of βPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front–rear cell polarity and directional cell migration.     

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.     

Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites

Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.     

Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cgamma phosphorylation

Vascular endothelial cell growth factor-A(165) (VEGF-A(165)) is critical for angiogenesis. Although protein kinase C-mediated protein kinase D(PKD)activation was implicated in the response, the detailed mechanism remains unclear. In this study, we found that VEGF-A(165)-stimulated tyrosine phosphorylation of PKD and the dominant negative mutant of PKD, PKD(Y463F), inhibited VEGF-A(165)-induced human umbilical vein endothelial cell (HUVEC) proliferation. In addition, PKD(S738A/S742A) overexpression inhibited VEGF-induced HUVEC migration. Furthermore, knockdown of PKD by its specific small interfering RNA inhibited VEGF-induced HUVEC proliferation and migration. Moreover transfection of PKD(Y463F), PKD(S738A/S742A), or PKD-small interfering RNA blocked VEGF-induced angiogenesis in vivo. Our signaling experiments show that KDR not Flt-1 mediated PKD tyrosine phosphorylation and KDR tyrosine residues 951 and 1059 were required for VEGF-A(165)-stimulated PKD serine and tyrosine phosphorylation, respectively. Whereas G protein Gbetagamma subunits were required for both PKD serine phosphorylation and tyrosine phosphorylation, intracellular Ca(2+) mobilization was required for VEGF-A(165)-stimulated PKD tyrosine phosphorylation and phospholipase C (PLC) activity was required for PKD serine phosphorylation. Surprisingly, the PLC inhibitor did not inhibit PKD tyrosine phosphorylation. Instead, PKD tyrosine 463 was required for VEGF-A(165)-stimulated PLCgamma tyrosine phosphorylation. Moreover, PKD interacted with PLCgamma even in unstimulated cells, and PKD tyrosine 463 phosphorylation was not required for this interaction. Together, we demonstrate that PKD interacts with PLCgamma and becomes tyrosine phosphorylated upon VEGF stimulation, leading to PLCgamma activation and angiogenic response of VEGF-A(165).     

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide

Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect.