p130cas but not paxillin is essential for Caco-2 intestinal epithelial cell spreading and migration on collagen IV

We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.     

Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation

We have previously shown that in a HEK-293 cell line that overexpresses the C1a isoform of the calcitonin receptor (C1a-HEK), calcitonin induces the tyrosine phosphorylation of the focal adhesion-associated proteins HEF1 (a p130(Cas)-like docking protein), paxillin, and focal adhesion kinase and that it also stimulates the phosphorylation and activation of Erk1 and Erk2. We report here that cell attachment to the extracellular matrix, an intact actin cytoskeleton, and c-Src are absolutely required for the calcitonin-induced phosphorylation of focal adhesion-associated proteins. In contrast to the phosphorylation of paxillin and HEF1 in cells attached to fibronectin-coated dishes, calcitonin failed to stimulate the phosphorylation of paxillin and HEF1 in suspended cells, in cells attached to poly-d-lysine-coated dishes, and in attached cells pretreated with the RGD-containing peptide GRGDS. Overexpression of wild-type c-Src increased calcitonin-induced paxillin and HEF1 phosphorylation, whereas overexpression of kinase-dead Src or Src lacking a functional SH2 domain inhibited the calcitonin-stimulated tyrosine phosphorylation of these proteins. Overexpression of Src lacking the SH3 domain did not affect the calcitonin-induced phosphorylation of paxillin and HEF1. In contrast to the regulation of paxillin and HEF1 phosphorylation, the calcitonin-induced phosphorylation of Erk1 and Erk2 did not appear to involve c-Src and was only partially dependent on cell adhesion to the extracellular matrix and an intact actin cytoskeleton. Furthermore, inhibition of Erk1 and Erk2 phosphorylation had no effect on the calcitonin-induced phosphorylation of paxillin and HEF1. Thus, in C1a-HEK cells, the calcitonin receptor is coupled to the tyrosine phosphorylation of focal adhesion-associated proteins and to Erk1/2 phosphorylation by mechanisms that are in large part independent.     

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.     

Focal adhesion targeting of v-Crk is essential for FAK phosphorylation and cell migration in mouse embryo fibroblasts deficient src family kinases or p130CAS

We examined the consequences of v-Crk expression in mouse embryo fibroblasts deficient Src family kinases or p130CAS. We found that Src kinases are essential for p130CAS/v-Crk signaling leading to FAK phosphorylation and cell migration in which Src is likely to mediate the focal adhesion targeting of v-Crk. SYF cells showed only low levels of FAK phosphorylation and cell migration, even in the presence of v-Crk. Expression of v-Crk restored migration of p130CAS-deficient cells to the level of wild-type cells, most likely through the targeting of v-Crk to focal adhesions by cSrc. In addition, we identified a new v-Crk-interacting protein that mediates v-Crk signaling in p130CAS-deficient cells. Using RT-PCR and caspase cleavage assays, we confirmed that this protein is not p130CAS and is responsible for maintaining v-Crk/Src signaling and migration in these. These findings suggest that focal adhesion targeting of v-Crk is essential in v-Crk-mediated cellular signaling and that v-Crk must form a complex with p130CAS or a p130CAS substitute to transduce signaling from the extracellular matrix.     

Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130^CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

RACK1 regulates Src activity and modulates paxillin dynamics during cell migration

Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.     

Tac-beta1 inhibits FAK activation and Src signaling

The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin β1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-β1) inhibits cell spreading. To study the mechanism whereby Tac-β1 inhibits cell spreading, we examined the effect of Tac-β1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-β1 and found that Tac-β1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-β1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-β1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-β1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.     

Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells

Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.     

Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.     

AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation and breast cancer cell growth pattern

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.     

c-Src mediates mitogenic signals and associates with cytoskeletal proteins upon vascular endothelial growth factor stimulation in Kaposi's sarcoma cells

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi's sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.     

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.     

Protein-tyrosine kinase 6 promotes peripheral adhesion complex formation and cell migration by phosphorylating p130 CRK-associated substrate

Protein-tyrosine kinase 6 (PTK6) is a non-myristoylated intracellular tyrosine kinase evolutionarily related to Src kinases. Aberrant PTK6 expression and intracellular localization have been detected in human prostate tumors. In the PC3 prostate cancer cell line, the pool of endogenous activated PTK6, which is phosphorylated on tyrosine residue 342, is localized at the membrane. Expression of ectopic membrane-targeted PTK6 led to dramatic morphology changes and formation of peripheral adhesion complexes in PC3 cells. Peripheral adhesion complex formation was dependent upon PTK6 kinase activity. We demonstrated that p130 CRK-associated substrate (p130CAS) is a novel direct substrate of PTK6, and it works as a crucial adapter protein in inducing peripheral adhesion complexes. Activation of ERK5 downstream of p130CAS was indispensable for this process. Knockdown of endogenous PTK6 led to reduced cell migration and p130CAS phosphorylation, whereas knockdown of p130CAS attenuated oncogenic signaling induced by membrane-targeted PTK6, including ERK5 and AKT activation. Expression of membrane-targeted PTK6 promoted cell migration, which could be impaired by knockdown of p130CAS or ERK5. Our study reveals a novel function for PTK6 at the plasma membrane and suggests that the PTK6-p130CAS-ERK5 signaling cascade plays an important role in cancer cell migration and invasion.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration

Crk-associated substrate (p130(CAS) or CAS) is a major integrin-associated Src substrate that undergoes tyrosine phosphorylation at multiple YXXP motifs in its substrate domain (SD) to create docking sites for SH2-containing signaling effectors. Notably, recruitment of Crk adaptor proteins to the CAS SD sites is implicated in promoting cell migration. However, it is unclear which or how many of the 15 CAS SD YXXP tyrosines are critically involved. To gain a better understanding of CAS SD function, we assessed the signaling capacity of individual YXXP motifs. Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, we determined that the ten tyrosines in YXXP motifs 6-15 are the major sites of CAS SD phosphorylation by Src. Phosphopeptide binding assays showed that all of these sites are capable of binding the Crk SH2 domain. To evaluate the requirement for CAS YXXP sites in stimulating cell migration, a series of phenylalanine substitution variants were expressed in CAS -/- mouse embryo fibroblasts. CAS expression enhanced the rate of cell migration into a monolayer wound in a manner dependent on the major sites of Src phosphorylation. Effective wound healing was achieved by CAS variants containing as few as four of the major sites, indicating sufficiency of partial SD signaling function in this cell migration response.     

Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.     

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.