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Results for “Focal Adhesion Kinase 1”

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At least 19 recordsLinked to original sources

PubMed2002

MEK kinase 1 interacts with focal adhesion kinase and regulates insulin receptor substrate-1 expression.

MEK kinase 1 (MEKK1) has been shown to contribute to the regulation of cell migration, whereas focal adhesion kinase (FAK) is a major player involved in both cell migration and integrin signaling. Here we show that MEKK1 and FAK are co-immunoprecipitated from mouse fibroblasts. Moreover, the association between MEKK1 and FAK appears to be physiologically relevant, as it is enhanced by treatment with epidermal growth factor (EGF). Targeting FAK to the membrane also enhanced its association with MEKK1, indicating that MEKK1 is localized to a membrane-related subcellular domain, perhaps focal adhesions. Interestingly, the expression of insulin receptor substrate-1 (IRS-1) was diminished in MEKK1-deficient fibroblasts, which is similar to an earlier finding in FAK-deficient fibroblasts. Insulin-like growth factor 1 (IGF-1)-induced ERK activation was diminished in MEKK1-deficient cells, but phosphatidylinositol 3-kinase/Akt activation was not. Although integrin reportedly regulates the transcription of the IRS-1 gene via FAK-mediated JNK activation, no impairment of fibronectin-stimulated activation of FAK, ERK, or JNK was observed in MEKK1-deficient cells. Reconstitution of MEKK1 expression restored IRS-1 expression as well as IGF-1-induced ERK activation. Taken together, these findings indicate that MEKK1 interacts with FAK in focal adhesions and regulates IRS-1 expression.

Animals↗

PubMed2002

Possible involvement of the vascular endothelial growth factor-Flt-1-focal adhesion kinase pathway in chemotaxis and the cell proliferation of osteoclast precursor cells in arthritic joints.

Vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of inflammatory joint disease, including angiogenesis and synovitis. Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive synovitis and subsequent bone destruction mediated by osteoclasts (OCs). In this study, we investigate the effects of VEGF on OC precursor cells (pOCs) using Raw cells and adjuvant-induced arthritis in rats. OCs and pOCs in the arthritic joints express VEGF and VEGF receptor type I (Flt-1). Raw cells also express Flt-1, and VEGF treatment stimulated chemotaxis, cell proliferation, the association of Flt-1 with focal adhesion kinase (FAK), and the tyrosine phosphorylation of FAK in Raw cells. The tyrosine phosphorylation of FAK was also observed in pOCs in the arthritic joints of adjuvant-induced arthritis. Adenovirus-mediated expression of FAK-related nonkinase in Raw cells inhibited the effects of VEGF in a dominant negative manner. Furthermore, intra-articular injection of the FAK-related nonkinase virus suppressed the recruitment of pOCs and bone destruction. Our results suggest the possible involvement of the VEGF-Flt-1-FAK pathway in inflammatory disease-induced joint destruction.

Animals↗

PubMed2005

Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase.

Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194-260 as the shortest FAK-interacting domain among those tested. Domain 194-260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194-260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684-1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.

Animals↗

PubMed2001

Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase.

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1delta, but not alpha or gamma1, at focal adhesions. PP1delta also co-immunoprecipitated with the focal adhesion kinase (FAK) and the alphav-integrin. In the present study glutathione S-transferase (GST)-PP1delta pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of delta. The association was confirmed by the ability to GST-FAK-related non-kinase (FRNK) to pull-down PP1delta from fibroblasts extract. GST-FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2-cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2 h from the mitotic release (when 85-90% of the cells remained round) and decreased to basal level by 8 h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4 h. FAK obtained from cells at 1.5 h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8 h was PP1delta. The results suggest that FAK dephosphorylation by PP1delta occurs in cells released from mitosis, and confirmed the specific association of PP1delta, as detected previously in adherent cells.

Animals↗

PubMed2000

Association of beta 1 integrin with focal adhesion kinase and paxillin in differentiating Schwann cells.

Schwann cells (SCs) differentiate into a myelinating cell when simultaneously adhering to an axon destined for myelination and basal lamina. We are interested in defining the signaling pathway activated by basal lamina. Using SC/sensory neuron (N) cocultures, we identified beta1 integrin and F-actin as components of a pathway leading to myelin gene expression and myelination (Fernandez-Valle et al., 1994, 1997). Here, we show that focal adhesion kinase (FAK) and paxillin are constitutively expressed by SCs contacting axons in the absence of basal lamina. Tyrosine phosphorylation of FAK and paxillin increases as SCs form basal lamina and differentiate. FAK and paxillin specifically coimmunoprecipitate with beta1 integrin in differentiating SC/N cocultures but not SC-only cultures. Paxillin coimmunoprecipitates with FAK and fyn kinase in differentiating SC/N cocultures. A subset of tyrosine-phosphorylated beta1 integrin, FAK, and paxillin molecules reside in the insoluble, F-actin-rich fraction of differentiating cocultures. Cytochalasin D, an actin depolymerizing agent, decreases tyrosine phosphorylation of FAK and paxillin and their association with beta1 integrin and causes a dose-dependent increase in the abundance of insoluble FAK and paxillin complexes. Collectively, our work indicates that beta1 integrin, FAK, paxillin, and fyn kinase form an actin-associated complex in SCs adhering to basal lamina in the presence of axons. This complex may be important for initiating the process of SC differentiation into a myelinating cell.

Animals↗

PubMed1999

The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes.

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the beta2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the beta2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

Actins↗

PubMed2004

Nerve growth factor (NGF) induces a rapid and sustained downregulation of the focal adhesion kinase (FAK).

1. Exposure of PC12 cells to nerve growth factor (NGF) induces an early tyrosine phosphorylation of many proteins, a number of which is still unidentified. Although NGF is known to bind to and activate the receptor tyrosine kinase TrkA, many downstream targets of NGF signaling may be possibly phosphorylated by nonreceptor tyrosine kinases such as c-Src and focal adhesion kinase (FAK). 2. In the present study, exposure of TrkA-overexpressing PC12 cells to NGF is found to cause a rapid and sustained loss in the recovery of a subpopulation of nominally active FAK (i.e., being autophosphorylated on the positive site of regulation). 3. Consistent with the possibility that NGF induces the proteolysis of FAK via recruitment of Src family kinases, the use of various phosphorylation site-specific anti-FAK antibodies revealed an NGF-inducible and PP1-sensitive accumulation of a putative fragment (i.e., p62) of FAK. Significantly, the mitogenic epidermal growth factor (EGF) failed to induce the downregulation of FAK and the accumulation of tyrosine phosphorylated p62. Such differential response of FAK to NGF and EGF may shape the specificity by which these growth factors control the status of cell-matrix adhesion and the adhesion-driven signaling.

Animals↗

PubMed2001

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells.

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.

Animals↗

PubMed1997

Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase.

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase beta (CAKbeta) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFalpha and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH2-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

Animals↗

PubMed1999

T cell activation up-regulates the expression of the focal adhesion kinase Pyk2: opposing roles for the activation of protein kinase C and the increase in intracellular Ca2+.

T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca2+/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca2+ ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca2+ has opposing effects on IL-2 and Pyk2 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca2+ ionophore-mediated inhibition of Pyk2 expression, implicating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.

Amino Acid Sequence↗

PubMed1998

Urea and NaCl differentially regulate FAK and RAFTK/PYK2 in mIMCD3 renal medullary cells.

Two cytosolic tyrosine kinases, focal adhesion kinase (FAK) and the newly described FAK homolog, related adhesion focal tyrosine kinase (RAFTK, also called PYK2 and CAKbeta), have been implicated in signaling to multiple mitogen-activated protein kinase (MAPK) pathways. Therefore, the ability of NaCl and urea to activate these kinases was investigated by in vitro kinase assay and anti-phosphotyrosine immunoblotting. RAFTK was promptly but only transiently activated by urea (within 1 min; 45%), whereas NaCl activated this kinase at 1, 5, 15, and 30 min of treatment (35-60%). In contrast, FAK exhibited only subtle regulation by the two solutes; however, the time course of induction was distinct for each solute. NaCl activated FAK at 1, 5, and 15 min (25-40%), whereas urea-inducible FAK activation (30%) was not evident until fully 15 min of treatment. At 5 min of treatment with increasing concentrations of solute, both urea and NaCl activated RAFTK in a dose-dependent and comparable fashion, culminating in an approximately twofold activation at 800 mosmol/kgH2O solute. Consistent with these data, solute treatment also enhanced tyrosine phosphorylation of RAFTK.

Animals↗

PubMed2001

Signaling events following chemokine receptor ligation in human dendritic cells at different developmental stages.

Responsiveness of dendritic cells (DC) to inflammatory CC chemokines is down-regulated during their maturation. We analyzed the mechanism underlying these events. Cell-surface expression of CC chemokine receptor (CCR)-1, -3 and -5 was increased during differentiation of immature DC (iDC) from monocytes. In contrast, these expressions were decreased during development of iDC into mature DC (mDC) to levels similar to those of monocytes. Transcriptional expression of CCR-1, -3 and -5 was increased during differentiation of iDC from monocytes, while the expression was decreased during development of iDC into mDC. Expression of CCR-7 transcript was detected in mDC, but not in monocytes or iDC. Both monocytes and iDC, but not mDC, migrated in response to inflammatory CC chemokines such as regulated on activation normal T cell expressed and secreted (RANTES)/CCL5, whereas mDC, but not monocytes or iDC, migrated to macrophage inflammatory protein (MIP)-3ss/CCL19. Receptor engagement of monocytes or iDC by RANTES (for CCR-1, -3 and -5) resulted in protein tyrosine phosphorylation events including activation of focal adhesion kinase as well as mitogen-activated protein kinase, whereas this stimulation induced little activation of these molecular events in mDC when compared with monocytes or iDC. On the other hand, stimulation with MIP-3ss (for CCR-7) induced tyrosine phosphorylation events in mDC, but not in monocytes or iDC. These results suggest that the down-regulation of cell-surface expression of CCR and of their downstream signaling events may be involved in the reduced chemotaxis of DC to inflammatory CC chemokines during their maturation.

Cell Differentiation↗

PubMed2004

Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells.

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

Cell Differentiation↗

PubMed1998

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle.

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

Actins↗

PubMed2000

Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways.

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.

Animals↗

PubMed1998

Focal adhesion kinase pp125FAK and the beta 1 integrin subunit are constitutively complexed in HaCaT cells.

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125FAK, an adhesion involved tyrosine kinase, with the integrin beta 1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible beta 1 integrin/pp125FAK complex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin beta 1 subunit is associated with tyrosine kinase pp125FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125FAK led to a reduced substrate adhesion.

Carcinoma, Squamous Cell↗

PubMed1995

Focal adhesion kinase (pp125FAK) is tyrosine phosphorylated after engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins on human T-lymphoblastic cells.

pp125FAK is a novel protein tyrosine kinase associated with focal adhesions. It has been shown that ligation of VLA beta 1 integrins on a number of cell types enhanced tyrosine phosphorylation and kinase activity of pp125FAK. Cellular transformation by retroviral oncogene products or mitogenic neuropeptides also result in the activation of this kinase. On the basis of these observations, pp125FAK has been proposed to be a key regulatory molecule connecting cell adhesion, transformation, and growth. We have previously shown that ligation of VLA beta 1 integrins induced CD3-dependent T cell proliferation and stimulated tyrosine phosphorylation of a molecular mass with a 105-kDa protein (pp105). Here we report that engagement of alpha 4 beta 1 and alpha 5 beta 1 integrins by adherence to their respective ligands stimulated tyrosine phosphorylation of 105- to 120-kDa proteins (pp105 and pp120, respectively) in human H9 T-lymphoblastic cells. At least one component of the 105- to 120-kDa proteins was found to be tyrosine-phosphorylated pp125FAK. While kinetics of adherence-dependent tyrosine phosphorylation of pp120/pp125FAK and pp105 are closely similar, pp105 appeared to be distinct from pp125FAK. Given T cell costimulation induced by VLA beta 1 integrins and the putative functional role of pp125FAK in cell growth, tyrosine phosphorylation of these two distinct proteins may be involved in T cell activation and proliferation.

Cell Adhesion↗