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.     

CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.     

Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.     

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.     

Tyrosine Y189 in the Substrate Domain of the Adhesion Docking Protein NEDD9 Is Conserved with p130Cas Y253 and Regulates NEDD9-Mediated Migration and Focal Adhesion Dynamics

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.     

Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas

We previously showed that activation of the small GTPase Cdc42 promotes breast cell migration on a collagen matrix. Here we further define the signaling pathways that drive this response and show that Cdc42-mediated migration relies on the adaptor molecule p130(Cas). Activated Cdc42 enhanced p130(Cas) phosphorylation and its binding to Crk. Cdc42-driven migration and p130(Cas) phosphorylation were dependent on the Cdc42 effector Ack1 (activated Cdc42-associated kinase). Ack1 formed a signaling complex that also included Cdc42, p130(Cas), and Crk, formation of which was regulated by collagen stimulation. The interaction between Ack1 and p130(Cas) occurred through their respective SH3 domains, while the substrate domain of p130(Cas) was the major site of Ack1-dependent phosphorylation. Signaling through this complex is functionally relevant, because treatment with either p130(Cas) or Ack1 siRNA blocked Cdc42-induced migration. These results suggest that Cdc42 exerts its effects on cell migration in part through its effector Ack1, which regulates p130(Cas) signaling.     

p130Cas controls the susceptibility of cancer cells to TGF-β-induced growth inhibition

Transforming growth factor-beta (TGF-β) suppresses the initiation of tumorigenesis by causing arrest at the G1 phase of the cell cycle. The loss of the antiproliferative function of TGF-β is a hallmark of many cancers. Here we report that p130Cas plays a role in determining the cellular responsiveness to TGF-β-induced growth inhibition in some cancer cells. An analysis of the tyrosine phosphorylation levels of p130Cas revealed higher levels of phosphorylation in cancer cell lines (MCF7 and A375) than in corresponding normal cell lines (MCF10A and MEL-STV). In contrast to normal cells, the cancer cells showed resistance to not only TGF-β-induced Smad3 phosphorylation and p21 expression, but also growth inhibition. However, silencing p130Cas using siRNA was sufficient to restore Smad3 phosphorylation and p21 expression, as well as the susceptibility to TGF-β-induced growth inhibition. Interestingly, the stable overexpression of p130Cas accelerated TGF-β-induced epithelial–mesenchymal transition. Our results suggest that elevated expression and tyrosine phosphorylation of p130Cas contributes to the resistance to TGF-β-induced growth inhibition, and thus to the initiation and progression of human cancers that harbor an active integrin signal.     

p130Cas: A key signalling node in health and disease

p130Cas/breast cancer anti-oestrogen resistance 1 (BCAR1) is a member of the Cas (Crk-associated substrate) family of adaptor proteins, which have emerged as key signalling nodes capable of interactions with multiple proteins, with important regulatory roles in normal and pathological cell function. The Cas family of proteins is characterised by the presence of multiple conserved motifs for protein–protein interactions, and by extensive tyrosine and serine phosphorylations. Recent studies show that p130Cas contributes to migration, cell cycle control and apoptosis. p130Cas is essential during early embryogenesis, with a critical role in cardiovascular development. Furthermore, p130Cas has been reported to be involved in the development and progression of several human cancers. p130Cas is able to perform roles in multiple processes due to its capacity to regulate a diverse array of signalling pathways, transducing signals from growth factor receptor tyrosine kinases, non-receptor tyrosine kinases, and integrins. In this review we summarise the current understanding of the structure, function, and regulation of p130Cas, and discuss the importance of p130Cas in both physiological and pathophysiological settings, with a focus on the cardiovascular system and cancer.     

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.     

Anchorage-independent phosphorylation of p130(Cas) protects lung adenocarcinoma cells from anoikis

The regulation and function of the signaling adaptor protein p130(Cas) in tumor cell anchorage-independent survival, or anoikis resistance, were investigated in human lung adenocarcinoma cells. The tyrosine phosphorylation and function of p130(Cas) during cell detachment were analyzed in tumor cells and compared with that of normal epithelial cells. Cell detachment trigged rapid dephosphorylation of p130(Cas) in the nontumorigenic and anoikis-sensitive normal epithelial cells, but had no effect on the tyrosine phosphorylation of p130(Cas) in the anoikis-resistant lung adenocarcinoma cells. Further analysis revealed that the total tyrosine kinase activities associated with p130(Cas) in the lung tumor cells are anchorage-independent and are significantly higher than that in the normal cells, in which the p130(Cas)-associated tyrosine kinase activities are anchorage-dependent. Analysis of two known p130(Cas)-associated tyrosine kinases FAK and Src indicated that the regulation of tyrosine phosphorylation of FAK and Src are altered in the tumor cells. Inhibition of Src specifically abolished phosphorylation of p130(Cas) and induced anoikis. Furthermore, overexpression of dominant-negative forms of p130(Cas) also induced apoptosis. Taken together, these data suggest that p130(Cas) mediates a cell survival signal from cell-matrix interaction. Alterations in tumor cells that lead to constitutive phosphorylation of p130(Cas) can prevent cells from anoikis, hence contribute to tumor cell anchorage independence and metastasis.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.     

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.     

Cooperative roles of Fyn and cortactin in cell migration of metastatic murine melanoma

Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.     

E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.     

The interaction of CFLAR with p130Cas promotes cell migration

CASP8 and FADD Like Apoptosis Regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis mediated by Fas and TRAIL. In addition to its anti-apoptotic function, CFLAR is also an important mediator of tumor growth. High level of CFLAR expression correlates with a more aggressive tumor. However, the mechanism of CFLAR signaling in malignant progression is not clear. Here we report a novel CFLAR-associated protein p130Cas, which is a general regulator of cell growth and cell migration. CFLAR-p130Cas association is mediated by the DED domain of CFLAR and the SD domain of p130Cas. Immunofluorescence observation showed that CFLAR had the colocalization with p130Cas at the focal adhesion of cell membrane. CFLAR overexpression promoted p130Cas phosphorylation and the formation of focal adhesion complex. Moreover, the enhancement of cell migration induced by CFLAR overexpression was obviously inhibited by p130Cas siRNA. In silico analysis on human database suggests high expressions of CFLAR or/and p130Cas are associated with poor prognosis of patients with lung cancer. Together, our results suggest a new mechanism for CFLAR involved in tumor development via association with p130Cas.     

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.     

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125(FAK), p130(Cas), and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK(A) receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK(A) receptor-mediated increase in p125(FAK), p130(Cas), and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.