Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

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).     

Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex

We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.     

Integrin activation and focal complex formation in cardiac hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.     

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.     

Requirements for the localization of p130 Cas to Z-lines in cardiac myocytes

The vertebrate heart responds to hemodynamic load with the enlargement of postmitotic, terminally differentiated cardiac myocytes. Such hypertrophic changes are characterized by alterations in sarcomeric organization and gene expression. Previously, we established a role for a nonreceptor tyrosine kinase, focal adhesion kinase, in signaling the changes in cytoskeletal organization associated with hypertrophy. Here, we report on data supporting a key role for p130Cas in this process. In neonatal cardiac myocytes FAK, Cas and paxillin are located in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. The expression of different Cas mutants results in a nearly complete loss of sarcomeric organization in these myocytes. Moreover, expression of the C-terminal focal adhesion-targeting domain of FAK both disrupted sarcomeric organization and interfered with the localization of endogenous Cas to Z-lines. These findings suggest that the association of FAK and Cas and the preservation of multiple protein-interaction motifs of Cas are required for the correct assembly of sarcomeres in cardiac myocytes.     

PTEN对SMMC—7721人肝癌细胞迁移增殖及粘着斑激酶磷酸化水平的影响

抑癌基因PTEN编码产物具有双专一性磷酸酶活性 ,并与细胞骨架张力蛋白同源。PTEN可参与粘着斑的形成和解聚而影响细胞迁移。现用PTEN表达质粒转染SMMC 772 1肝癌细胞 ,研究SMMC 772 1细胞运动能力的变化及PTEN与粘着斑激酶 (FAK)酪氨酸磷酸化水平之间的关系。PTEN过表达能够显著抑制细胞在Fn基质上的活动 :细胞在Fn基质上的迁移下降了 35 % ;在 30min和 6 0min两个时间点 ,Fn基质上细胞铺展分别降低了 2 9%和 2 6 % ;而在多聚赖氨酸基质上细胞铺展并没有变化。运用免疫沉淀和Western印迹方法 ,分析FAK及其酪氨酸磷酸化水平 ,发现PTEN过表达不影响FAK表达 ,但显著降低Fn诱导的FAK酪氨酸磷酸化水平 ,两者水平呈负相关。流式细胞仪分析细胞周期结果表明 ,PTEN抑制细胞 ,S期细胞下降了 16 %。上述结果提示 ,PTEN抑制肝癌细胞迁移铺展和增殖 ;PTEN对细胞运动的影响可能通过调节FAK酪氨酸磷酸化水平而实现。     

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.     

Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy

Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

p130Cas-dependent actin remodelling regulates myogenic differentiation

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Mechanotransduction and focal adhesions

Cellular FAs (focal adhesions) respond to internal and external mechanical stresses which make them prime candidates for mechanotransduction. Recent observations showed that the FA proteins including vinculin, FAK (FA kinase) and p130Cas are crucial for the ability of cells to transmit forces and to generate cytoskeletal tension. When mechanically stimulated, cells respond by modulating the spreading area, remodel the actin cytoskeleton, activate actomyosin interactions, recruit integrins and reinforce FAs and cytoskeletal structures. These complex cellular responses are orchestrated such that mechanical stresses within the FA complex remained within a narrow range.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase

In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions.     

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.     

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.     

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.     

Tyrosine dephosphorylation, but not phosphorylation, of p130Cas is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: implication of p130Cas involvement in pathways unrelated to cytoskeletal reorganization

The newly described adapter molecule p130 Crk-associated substrate (Cas) has been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncogene-mediated transformation. We characterized the regulation of Cas tyrosine phosphorylation in highly differentiated, anucleate platelets. Phospholipase C-activating receptor agonists, including collagen, thrombin receptor-activating peptide (TRAP), and U46619 (a thromboxane A2 analogue), and A23187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platelets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol, protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylation, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated in a manner dependent on integrin alpha IIb beta 3-mediated aggregation. While BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged it. Under the conditions, this PKC inhibitor suppressed platelet aggregation but not intracellular Ca2+ mobilization. In contrast to Cas involvement in focal adhesions in other cells, platelet Cas phosphorylation preceded the activation of focal adhesion kinase (FAK), and blockage of alpha IIb beta 3-mediated platelet aggregation with a GRGDS peptide resulted in prolongation of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylation was insensitive to cytochalasin D, an actin polymerization inhibitor. The failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activation. Of the signaling molecules investigated in this study, Src seemed to associate with Cas. Finally, Cas existed mainly in cytosol and membrane cytoskeleton fractions in the resting state, and remained unchanged during platelet aggregation, when FAK translocated to the cytoskeletal fraction. Our findings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation occurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, by itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine dephosphorylation, but not phosphorylation, is dependent on integrin alpha IIb beta 3-mediated aggregation; and (iv) Cas is not involved in cytoskeletal reorganization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.