A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions

c-Cbl-associated protein, CAP, was originally cloned from a 3T3-L1 adipocyte cDNA expression library using full-length c-Cbl as a bait. CAP contains a unique structure, with three adjacent Src homology-3 (SH3) domains in the COOH terminus and a region sharing significant sequence similarity with the peptide hormone sorbin. Expression of CAP in NIH-3T3 cells overexpressing the insulin receptor induced the formation of stress fibers and focal adhesions. This effect of CAP expression on the organization of the actin-based cytoskeleton was independent of the type of integrin receptors engaged with extracellular matrix, whereas membrane ruffling and decreased actin stress fibers induced by insulin were not affected by expression of CAP. Immunofluorescence microscopy demonstrated that CAP colocalized with actin stress fibers. Moreover, CAP interacted with the focal adhesion kinase, p125FAK, both in vitro and in vivo through one of the SH3 domains of CAP. The increased formation of stress fibers and focal adhesions in CAP-expressing cells was correlated with decreased tyrosine phosphorylation of p125FAK in growing cells or upon integrin-mediated cell adhesion. These results suggest that CAP may mediate signals for the formation of stress fibers and focal adhesions.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.     

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.     

Integrin alpha IIb beta 3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase

Activation of the focal adhesion kinase pp125FAK correlates with its phosphorylation on tyrosine residues and is mediated by multiple receptor-ligand pairs. In platelets, pp125FAK phosphorylation is triggered by alpha IIb beta 3 integrin or Fc gamma RII receptor interaction with immobilized fibrinogen and IgG, respectively. In this study we used platelets as a model system to explore the role of PI 3-kinase relative to pp125FAK phosphorylation. Treatment of the platelets with two PI 3-kinase inhibitors, wortmannin and LY294002, inhibited in a dose-dependent manner alpha IIb beta 3-mediated platelet spreading on fibrinogen having no effect on platelet spreading on IgG. Both inhibitors also completely abolished alpha IIb beta 3-mediated pp125FAK phosphorylation but not pp72syk phosphorylation. Furthermore, Fc gamma RII- and thrombin-induced pp125FAK phosphorylation were not affected by wortmannin and LY294002. Finally, the PI 3-kinase inhibitors' effect on alpha IIb beta 3-mediated spreading and pp125FAK phosphorylation was reversed by phorbol ester treatment. These results establish that the role of PI 3-kinase relative to pp125FAK phosphorylation in platelets is receptor type-specific yet essential for alpha IIb beta 3-mediated cell spreading and pp125FAK phosphorylation.     

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21rho, and the integrity of the actin cytoskeleton

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.     

Integrin-mediated signals regulated by members of the rho family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.     

Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by the immunosuppressants FK506 and cyclosporin A

We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, and was inhibited by monoclonal antibodies for integrins alpha2 and beta1. Indirect immunofluorescence microscopic observations revealed that integrin alpha2 and beta1 molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.     

PKC regulation of microfilament network organization in keratinocytes defined by a pharmacological study with PKC activators and inhibitors

The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mM genistein and 1 microM herbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 microM concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.     

Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1

BACKGROUND:. Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS:. Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS:. Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.     

Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent and -independent pathways

Endothelin-1 (ET-1) has been shown to induce DNA synthesis in primary astrocytes by stimulating the extracellular signal-regulated kinase (ERK) pathway. To clarify the mechanisms responsible for the anchorage-dependent growth of astrocytes, the relationships between cell adhesion and ERK activation were investigated. Here it is reported that ET-1 promotes the formation of stress fibers and focal adhesions and the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as Src activation and association of phosphorylated FAK with Grb2. Pretreatment of astrocytes with cytochalasin D or C3-transferase, which inhibits actin polymerization or Rho activity, respectively, prevented the activation/phosphorylation of Src, FAK, and paxillin after ET-1 stimulation; by contrast, the ERK pathway was not significantly affected. This differential activation of FAK/Src and ERK pathways was also observed with astrocytes 10 and 60 min after replating on poly-L-ornithine-precoated dishes. Collectively, these findings indicate that activation of FAK and Src is dependent on actin cytoskeleton integrity, Rho activation, and adhesion to extracellular matrix, whereas ERK activation is independent of these intracellular events and seems to correlate with activation of the newly identified protein tyrosine kinase PYK2. Induction of DNA synthesis by ET-1, however, was reduced dramatically in astrocytes pretreated with either cytochalasin D or C3-transferase. This study provides a demonstration of Rho- and adhesion-dependent activation of FAK/Src, which collaborates with adhesion-independent activation of PYK2/ERK for DNA synthesis in ET-1-stimulated astrocytes.     

Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin. Correlation with tyrosine phosphorylations of paxillin and FAK

To examine the role of C-terminal Src kinase (Csk), a negative regulatory kinase of Src family tyrosine kinases, in the cell adhesion mechanism of the nervous system, wild-type Csk (Csk), and a kinase-deficient mutant of Csk (Csk-DeltaK) were overexpressed in primary cultured type I astrocytes by infecting them with the recombinant adenovirus. Overexpression of Csk repressed the in vitro kinase activity of Src to as little as 10% that of control cells and interfered with cell spreading and cell attachment to fibronectin. Focal adhesion assembly and the organization of actin stress fibers were also disrupted in cells overexpressing Csk. On the other hand, overexpression of Csk-DeltaK induced tyrosine phosphorylation of cellular proteins, including the paxillin and focal adhesion kinase (FAK) and enhanced to some extent the cytoskeletal organization and the rate of cell spreading on fibronectin, indicating that Src or its relatives was functionally activated in the cells. Paxillin was also tyrosine-phosphorylated in Csk-overexpressing cells, indicating that it can serve as a substrate of Csk. The phosphorylation state of paxillin in cells overexpressing Csk was indistinguishable from that in cells expressing Csk-DeltaK in that both phosphorylated paxillins bound equally to SH2 domain of Csk and were co-immunoprecipitated with Csk. In contrast, tyrosine phosphorylation of FAK and its in vitro autophosphorylation activity were increased only in cells expressing Csk-DeltaK. In Csk-expressing cells, the kinase activity of FAK was substantially decreased to 20-30% that of control cells, even though the expression level of FAK was rather increased. These findings suggest that Csk regulates Src family tyrosine kinases that play essential roles in the regulation of cell adhesion via a FAK-dependent mechanism and that the tyrosine phosphorylation of paxillin alone may not be sufficient for the regulation of the cell adhesion mechanism in astrocytes.     

Outside-In signaling of soluble and solid-phase fibrinogen through integrin alphaIIbbeta3 is different and cooperative with each other in a megakaryoblastic leukemia cell line, CMK

The function and the outside-in signaling pathways of alphaIIbbeta3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of alphaIIbbeta3. Binding of soluble fibrinogen to the cells via alphaIIbbeta3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by alphaIIbbeta3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin beta3 and a complex formation of integrin beta3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways.     

Requirement for phosphatidylinositol 3'-kinase activity in platelet-derived growth factor-stimulated tyrosine phosphorylation of p125 focal adhesion kinase and paxillin

The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF-stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr-740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3'-kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin.     

Na-H exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading

The ubiquitously expressed Na-H exchanger NHE1 functions in regulating intracellular pH and cell volume. NHE1 activity is stimulated by hormones, growth factors, and activation of integrin receptors. We recently determined that NHE1 activity is also stimulated by activation of the low molecular weight GTPase RhoA and that increases in NHE1 activity are necessary for RhoA-induced formation of actin stress fibers. We now show that NHE1 acts downstream of RhoA to modulate initial steps in integrin signaling for the assembly of focal adhesions. Adhesion of CCL39 fibroblasts on fibronectin was markedly delayed in the presence of the NHE inhibitor ethylisopropylamiloride. In mutant PS120 cells, derived from CCL39 fibroblasts but lacking NHE1, adhesion was also delayed but was rescued in PS120 cells stably expressing NHE1. In the absence of NHE1 activity, cell spreading was inhibited, and the accumulation of integrins, paxillin, and vinculin at focal contacts was impaired. Additionally, tyrosine phosphorylation of p125(FAK) induced by integrin clustering was also impaired. Inactivation of RhoA with C3 transferase and inhibition of the Rho-kinase p160ROCK with the pyridine derivative Y-27632 completely abolished activation of NHE1 by integrins but not by platelet-derived growth factor. These findings indicate that NHE1 acts downstream of RhoA to contribute a previously unrecognized critical signal to proximal events in integrin-induced cytoskeletal reorganization.     

Low density lipoprotein phosphorylates the focal adhesion-associated kinase p125(FAK) in human platelets independent of integrin alphaIIb beta3

Low density lipoprotein (LDL) is known to sensitize platelets to agonists via integrin mediated outside-in signaling (Hackeng, C. M., Huigsloot, M., Pladet, M. W., Nieuwenhuis, H. K., Rijn, H. J. M. v., and Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc. Biol., in press). As outside in signaling is associated with phosphorylation of p125(FAK), the effect of LDL on p125(FAK) phosphorylation in platelets was investigated. LDL induced p125(FAK) phosphorylation in a dose- and time- dependent manner. The phosphorylation was independent of ligand binding to integrin alphaIIbbeta3 and aggregation, such in contrast to alpha-thrombin-induced p125(FAK) phosphorylation, that critically depended on platelet aggregation. Platelets from patients with Glanzmann's thrombastenia showed the same LDL- induced phos- phorylation of p125(FAK) as control platelets, whereas alpha-thrombin completely failed to phosphorylate the kinase in the patients platelets. LDL signaling to p125(FAK) was independent of integrin alpha2 beta1, the FcgammaRII receptor, and the lysophosphatidic acid receptor and not affected by inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or p38(MAPK). Phosphorylation of p125(FAK) by LDL was strongly inhibited by cyclic AMP. These observations indicate that LDL is a unique platelet agonist, as it phosphorylates p125(FAK) in platelet suspensions, under unstirred conditions and independent of integrin alphaIIb beta3.     

Gbeta gamma-independent coupling of alpha2-adrenergic receptor to p21(rhoA) in preadipocytes

In preadipocytes, alpha2-adrenergic receptor (alpha2-AR) stimulation leads to a Gi/Go-dependent rearrangement of actin cytoskeleton. This is characterized by a rapid cell spreading, the formation of actin stress fibers, and the increase in tyrosyl phosphorylation of the focal adhesion kinase (pp125(FAK)). These cellular events being tightly controlled by the small GTPase p21(rhoA), the existence of a Gi/Go-dependent coupling of alpha2-AR to p21(rhoA) in preadipocytes was proposed. In alpha2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line and which stably expresses the human alpha2C10-adrenergic receptor) alpha2-adrenergic-dependent induction of cell spreading, formation of actin stress fibers, and increase in tyrosyl phosphorylation of pp125(FAK) were abolished by pretreatment of the preadipocytes with the C3 exoenzyme, a toxin which impairs p21(rhoA) activity by ADP-ribosylation. Conversely, C3 exoenzyme had no effect on the alpha2-adrenergic-dependent increase in tyrosyl phosphorylation and shift of ERK2 mitogen-activated protein kinase. alpha2-Adrenergic stimulation also led to an increase in GDP/GTP exchange on p21(rhoA), as well as to an increase in the amount of p21(rhoA) in the particulate fraction of alpha2AF2 preadipocytes. Stable transfection of alpha2AF2 preadipocytes with the COOH-terminal domain of betaARK1 (betaARK-CT) (a blocker of Gbeta gamma-action), strongly inhibited the alpha2-adrenergic-dependent increase in tyrosyl phos- phorylation and shift of ERK2, without modification of the tyrosyl phosphorylation of pp125(FAK) and spreading of preadipocytes. These results show that alpha2-adrenergic-dependent reorganization of actin cytoskeleton requires the activation of p21(rhoA) in preadipocytes. Conversely to the activation of the p21(ras)/mitogen-activated protein kinase pathway, the alpha2-adrenergic activation of p21(rhoA)-dependent pathways are independent of the beta gamma-subunits of heterotrimeric G proteins.     

Intravascular MR imaging of atherosclerotic plaque: ex vivo analysis of human femoral arteries with histologic correlation

Protein tyrosine phosphorylation was examined on a human glioblastoma cell line, T98G, after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of a 125 kDa protein at 30 min after stimulation. The 125-kDa molecule phosphorylated was revealed to be a focal adhesion kinase (FAK). Tyrosine phosphorylation of p125FAK continued at least up to 5 h, and decreased after 8 h concomitant with apoptosis. Tyrosine phosphorylation of p125FAK was blocked by herbimycin A, a potent inhibitor of protein tyrosine kinases, while apoptosis was accelerated. When T98G cells were incubated with FAK antisense oligonucleotide, apoptosis was also accelerated. These results suggest that tyrosine phosphorylation of p125FAK plays a suppressive role in hydrogen peroxide-induced apoptosis.     

Galpha12 and Galpha13 stimulate Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate

We examined whether constitutively active mutants of the Galpha proteins Galpha12 and Galpha13, which together comprise the G12 subfamily of Galpha proteins, induce Rho-dependent tyrosine phosphorylation of the focal adhesion proteins p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate. We report that transient expression of the constitutively active mutants of Galpha12 or of Galpha13 in human embryonic kidney 293 cells stimulates tyrosine phosphorylation of a set of proteins of Mr of 110,000-130,000, 97,000, and 60,000-70,000. We identified p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate as prominent tyrosine-phosphorylated proteins in human embryonic kidney 293 cells expressing constitutively active Galpha12 and Galpha13. In common with the increased tyrosine phosphorylation of these proteins mediated by mitogens acting through heptahelical receptors, the Galpha12- and Galpha13-mediated increase in tyrosine phosphorylation is blocked by cytochalasin D, which specifically disrupts the actin cytoskeleton, and by the Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Our results support the hypothesis that Galpha12 and Galpha13 activate Rho and suggest that Galpha12 and Galpha13 may mediate the tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate.