PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK,Cas, and paxillin,formation of Cas-CRK complex,and JNK activation

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.     

Role of c-Abl in directing metabolic versus mitogenic effects in insulin receptor signaling

c-Abl is a cytoplasmic tyrosine kinase involved in several signal transduction pathways. Here we report that c-Abl is involved also in insulin receptor signaling. Indeed, c-Abl tyrosine kinase is activated upon insulin stimulation. Inhibition of c-Abl tyrosine kinase by STI571 attenuates the effect of insulin on Akt/GSK-3beta phosphorylation and glycogen synthesis, and at the same time, it enhances the effect of insulin on ERK activation, cell proliferation, and migration. This effect of STI571 is specific to c-Abl inhibition, because it does not occur in Abl-null cells and is restored in c-Abl-reconstituted cells. Numerous evidences suggest that focal adhesion kinase (FAK) is involved in mediating this c-Abl effect. First, anti-phosphotyrosine blots indicate that c-Abl tyrosine kinase activation is concomitant with FAK dephosphorylation in response to insulin, whereas c-Abl inhibition is accompanied by FAK phosphorylation in response to insulin, a response similar to that observed with IGF-I. Second, the c-Abl effects on insulin signaling are not observed in cells devoid of FAK (FAK(-/-) cells). Taken together these results suggest that c-Abl activation by insulin, via a modification of FAK response, may play an important role in directing mitogenic versus metabolic insulin receptor signaling.     

Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase,phosphatidylinositol 3-kinase and extracellular signal regulated kinases

Insulin plays an important role in muscle cell survival and proliferation. However, there is no report showing the role of insulin in spreading of muscle cells. In the present report, we showed that insulin enhances muscle cell spreading concomitant with enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Moreover, insulin can stimulate the cell spreading even in presence of integrin alpha5 blockers although to a lesser extent as compared to control. Cell adhesion was not dependent on insulin and serum, and decreased in presence of integrin blockers. We found direct association of FAK with affinity purified insulin receptors using in vitro kinase assay. The increase in FAK tyrosine phosphorylation was associated with increase in its kinase activity and further supported by increased phosphotyrosine accumulation on focal adhesions and increased membrane localization of FAK after stimulation by insulin. Moreover, insulin-mediated muscle cell spreading was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase) activity. PI 3-kinase activity was found to be associated with FAK and the FAK associated PI 3-kinase activity enhanced when cells were plated in presence of insulin. We also observed activation of MAP kinases, i.e., ERK-1/-2 during insulin mediated muscle cell spreading. In conclusion, FAK, PI 3-kinase, and MAP kinase are important components of pathway(s) that regulate insulin stimulated muscle cell spreading.     

Focal adhesion kinase negatively regulates neuronal insulin resistance

Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.     

Signaling between focal adhesion kinase and trio

The Trio guanine nucleotide exchange factor functions in neural development in Caenorhabditis elegans and Drosophila and in the development of neural tissues and skeletal muscle in mouse. The association of Trio with the Lar tyrosine phosphatase led us to study the role of tyrosine phosphorylation in Trio function using focal adhesion kinase (FAK). The Lar-interacting domain of Trio is constitutively tyrosine-phosphorylated when expressed in COS-7 cells and was highly phosphorylated when it was co-transfected with FAK. Co-precipitation studies indicated that Trio binds to the FAK amino-terminal domain and to the FAK kinase domain via its SH3 and kinase domains, respectively. Tyrosine-phosphorylated FAK and Trio were present mainly in the detergent-insoluble fraction of cell lysates, and co-expression of Trio and FAK resulted in increased amounts of Trio present in the detergent-insoluble fraction. Immunofluorescence of cells co-transfected with FAK and Trio revealed significant co-localization of the proteins at the cell periphery, indicating that they form a stable complex in vivo. A FAK phosphorylation site, tyrosine residue 2737, was identified in subdomain I of the Trio kinase domain. Additionally, in vitro phosphorylation assays and in vivo co-expression studies indicated that Trio enhances FAK kinase activity. These results suggest Trio may be involved in the regulation of focal adhesion dynamics in addition to effecting changes in the actin cytoskeleton through the activation of Rho family GTPases.     

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.     

Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.     

Role of focal adhesion kinase in MAP kinase activation by insulin-like growth factor-I or insulin.

Integrin-induced focal adhesion kinase (FAK) phosphorylation as well as insulin-like growth factor-I (IGF-I) and insulin activate MAP kinase. Since IGF-I or insulin have been suggested to affect FAK phosphorylation, we analyzed the role of FAK in IGF-I- or insulin-induced MAP kinase activation. Although MAP kinase was stimulated by IGF-I or insulin, FAK tyrosine phosphorylation remained unchanged in fibroblasts expressing normal or transiently elevated levels of IGF-I and insulin receptors. Further analysis in FAK deficient fibroblasts suggested that FAK impedes MAP kinase activation by IGF-I or insulin.     

Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle

Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.     

尾加压素增加血管平滑肌细胞粘着斑激酶的含量和活力

在体外培养的血管平滑肌细胞上,采用蛋白印迹杂交免疫沉淀的方法,研究尾加压素Ⅱ（ＵⅡ）与粘着斑激酶（ＦＡＫ）介导的信号传导之间的关系。结果表明,ＵⅡ在１０＾－８、１０＾－７和１０＾－６ｍｏｌ／Ｌ的深度时,可分别使ＦＡＫ活力增加２．２、３．０４和０．７３倍。加入ＵⅡ在（１０＾０７ｍｏｌ／Ｌ）５ｍｉｎ后,ＦＡＫ活力增加９５％,但与对照组相比无显著性差异（Ｐ〉０．０５０,刺激１０ｍｉｎ后,ＦＡＫ活力升高３     

Mitogen-induced,FAK-dependent tyrosine phosphorylation of the SSeCKS scaffolding protein

The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.     

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.     

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.     

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.     

Regulation of heterochromatin remodelling and myogenin expression during muscle differentiation by FAK interaction with MBD2

Focal adhesion kinase (FAK), a major cell adhesion‐activated tyrosine kinase, has an important function in cell adhesion and migration. Here, we report a new signalling of FAK in regulating chromatin remodelling by its interaction with MBD2 (methyl CpG‐binding protein 2), underlying FAK regulation of myogenin expression and muscle differentiation. FAK interacts with MBD2 in vitro, in myotubes, and in isolated muscle fibres. Such an interaction, increased in myotubes exposed to oxidative stress, enhances FAK nuclear localization. The nuclear FAK–MBD2 complexes alter heterochromatin reorganization and decrease MBD2 association with HDAC1 (histone deacetylase complex 1) and methyl CpG site in the myogenin promoter, thus, inducing myogenin expression. In line with this view are observations that blocking FAK nuclear localization by expressing dominant negative MBD2 or suppression of FAK expression by its miRNA in C2C12 cells attenuates myogenin induction and/or impairs muscle‐terminal differentiation. Together, these results suggest an earlier unrecognized role of FAK in regulating chromatin remodelling that is important for myogenin expression and muscle‐terminal differentiation, reveal a new mechanism of MBD2 regulation by FAK family tyrosine kinases, and provide a link between cell adhesion and chromatin remodelling.     

Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase

Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.