Kindlin-2 Tyrosine Phosphorylation and Interaction with Src Serve as a Regulatable Switch in the Integrin Outside-in Signaling Circuit

Integrin-mediated cell-extracellular matrix (ECM) adhesion is critical for control of intracellular signaling; however, the mechanisms underlying this “outside-in” signaling are incompletely understood. Here we show that depletion of kindlin-2 impairs integrin outside-in signaling. Kindlin-2 is tyrosine-phosphorylated upon cell-ECM adhesion. Furthermore, kindlin-2 binds Src in a cell-ECM adhesion-regulatable fashion. At the molecular level, the kindlin-2·Src interaction is mediated by the kindlin-2 F0 and the Src SH2 and SH3 domains. Src activation increases kindlin-2 tyrosine phosphorylation and the kindlin-2·Src interaction. Conversely, inhibition of Src reduces kindlin-2 tyrosine phosphorylation and diminishes the kindlin-2·Src interaction. Finally, disruption of the kindlin-2·Src interaction, unlike depletion of kindlin-2, impairs neither cell-ECM adhesion nor cell-ECM adhesion-induced focal adhesion kinase Tyr-397 phosphorylation. However, it markedly inhibits cell-ECM adhesion-induced paxillin tyrosine phosphorylation, cell migration, and proliferation. These results suggest that kindlin-2 tyrosine phosphorylation and interaction with Src serve as a regulatable switch downstream of focal adhesion kinase in the integrin outside-in signaling circuit, relaying signals from cell-ECM adhesion to paxillin that control cell migration and proliferation.     

COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

Platelet-derived growth factor inhibits smooth muscle cell adhesion to fibronectin by ERK-dependent and ERK-independent pathways

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

MicroRNAs are essential for stretch-induced vascular smooth muscle contractile differentiation via microRNA (miR)-145-dependent expression of L-type calcium channels

Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca(2+)/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.     

Integrin-induced tyrosine phosphorylation of protein-tyrosine phosphatase-alpha is required for cytoskeletal reorganization and cell migration

Protein-tyrosine phosphatase-alpha (PTPalpha) activates Src family kinases (SFKs) to promote the integrin-stimulated early autophosphorylation of focal adhesion kinase (FAK). We report here that integrin stimulation induces tyrosine phosphorylation of PTPalpha. PTPalpha was dephosphorylated upon fibroblast detachment from the substratum and rephosphorylated when cells were plated on the integrin ligand fibronectin. alpha PTP phosphorylation occurred at Tyr789 and required SFKs (Src or Fyn/Yes), FAK, and an intact cytoskeleton. It also required active PTPalpha or constitutively active Src. These observations indicate that PTPalpha activates SFKs and that the subsequently activated SFK.FAK tyrosine kinase complex in turn phosphorylates PTPalpha. Reintroduction of wild-type PTPalpha or unphosphorylatable PTPalpha(Y789F) (but not inactive PTPalpha) into PTPalpha-null fibroblasts restored defective integrin-induced SFK activation, FAK phosphorylation, and paxillin phosphorylation. PTPalpha(Y789F) and inactive PTPalpha could not rescue delayed actin stress fiber assembly and focal adhesion formation or defective cell migration. This study distinguishes two roles of PTPalpha in integrin signaling: an early role as an activator of SFKs and FAK with no requirement for PTPalpha phosphorylation and a later downstream role in cytoskeleton-associated events for which PTPalpha phosphorylation at Tyr789 is essential.     

Src modulates contractile vascular smooth muscle function via regulation of focal adhesions

Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissue's ability to contract, and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause and effect manner, regulates focal adhesion protein function and, consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signalling

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P?≤?0.001); PKC or Src inhibition also prevented focal adhesion formation. Western blotting demonstrated that during spreading and adhesion these inhibitors also impaired PKC and FAK activation, with Gö 6976 or SrcI inhibiting FAK phosphorylation by at least 70% (P?≤?0.001), and herbimycin A or SrcI inhibiting PKC phosphorylation by at least 46% (P?≤?0.01). Confocal microscopy revealed phosphorylated PKC colocalised with focal adhesion sites, particularly during early phases of adhesion and spreading. Finally, fibronectin promoted PKC and FAK phosphorylation in suspended haemocytes demonstrating that activation can occur independent of cell adhesion. These novel data are consistent with PKC and FAK/Src playing an integrated role in integrin activation and integrin-mediated spreading by L. stagnalis haemocytes. We propose a model in which integrin engagement mediates association of PKC with FAK/Src complexes to promote focal adhesion assembly during immune recognition by these cells.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

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

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

Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.     

Phosphorylation of Ser 21 in Fyn regulates its kinase activity,focal adhesion targeting,and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Ligation of HLA class I molecules on endothelial cells induces phosphorylation of Src,paxillin, and focal adhesion kinase in an actin-dependent manner

The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection.     

Focal adhesion kinase (FAK), a multifunctional protein

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase localized to regions called focal adhesions. Many stimuli can induce tyrosine phosphorylation and activation of FAK, including integrins and growth factors. The major site of autophosphorylation, tyrosine 397, is a docking site for the SH2 domains of Src family proteins. The other sites of phosphorylation are phosphorylated by Src kinases. Phosphorylated FAK binds proteins of focal adhesion and can activate them directly or indirectly by phosphorylation. These activated proteins forming the FAK complex facilitate the generation of downstream signals necessary to regulate cell functions, like motility, survival and proliferation. Dysregulation of FAK could participate in the development of cancer. This review will focus upon the mechanisms by which FAK transmits biochemical signals and elicits biological effects.     

Src tyrosine kinases mediate activations of NF-kappaB and integrin signal during lipopolysaccharide-induced acute lung injury

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-kappaB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-alpha and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-kappaB activation, and phosphorylation and degradation of IkappaB-alpha. The inhibition of NF-kappaB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin beta(3), and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-alpha(v), anti-beta(3), or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-kappaB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-kappaB and integrin (alpha(v)beta(3)) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.     

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

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