Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling pathways in fibroblasts

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.     

Integrins in morphogenesis and signaling

Integrins are a family of heterodimeric transmembrane receptors that provide a physical and biochemical bridge between components of the extracellular matrix and the intracellular physiological environment. Binding of integrins to their ligands results in the formation of cytoplasmic multi-protein assemblies composed of both cytoskeletal and signaling molecules. The composition and activity of these assemblies is regulated by the nature of integrin-ligand interactions, as well as by intracellular regulators that include tyrosine kinases and phosphatases, PKC, and small GTPases. Integrin-mediated cellular physiological responses include the activation of signal transduction, cytoskeletal rearrangements, and co-regulation of growth factor activities. These responses, combined with integrin-mediated cell adhesion, play a major role in tissue morphogenesis and developmental processes.     

Role of focal adhesion kinase in integrin signaling

Integrins are the major cell surface receptors for extracellular matrix molecules, which play critical roles in a variety of biological processes. Focal adhesion kinase has recently been established as a key component of the signal transduction pathways triggered by integrins. Aggregation of FAK with integrins and cytoskeletal proteins in focal contacts has been proposed to be responsible for FAK activation and autophosphorylation by integrins in cell adhesion. This may be achieved by FAK interaction with talin or other cytoskeletal proteins that in turn associate with the cytoplasmic domain of integrin beta subunits. Autophosphorylation of FAK at Y397 leads to its association with Src, resulting in activation of both kinases. The activated FAK/Src complex acts on potential substrates tensin, paxillin and p130cas. Besides cytoskeletal regulation, FAK phosphorylation and/or binding to paxillin and p130cas may trigger downstream activation of MAP kinase by the adoptor protein Crk. Src association with FAK may also lead to its phosphorylation of other sites on FAK, including a binding site for Grb2. Cell adhesion-dependent association of FAK and Grb2 may provide a mechanism by which MAP kinase is activated in cell adhesion. PI 3-kinase has also been shown to bind FAK in a cell adhesion-dependent manner at the major autophosphorylation site Y397. This association could lead to activation of PI 3-kinase and its downstream effectors. Recent results from a number of different approaches have shown that integrin signaling through FAK leads to increased cell migration on fibronectin as well as potentially regulating cell proliferation and survival.     

Defining the microbiological quality of dialysis fluid

Integrins are dynamic cell-surface receptors that provide a physical link between the extracellular matrix and the cell cytoskeleton. The interaction of integrins with extracellular matrix ligands also results in the generation of intracellular signals that regulate cell survival and growth, cell differentiation, and cell motility. The signaling pathways regulated by integrins are both diverse and complex, and involve cross-talk among multiple pathways. An added level of complexity resides in the findings that intracellular signals generated by a variety of agonists can modulate cell adhesion by regulating integrin-matrix interactions. The identification and functional characterization of integrin receptor-proximal components involved in this fascinating "inside-out" and "outside-in" signal transduction remains an area of intense investigation. This review focuses on recent advances in this area.     

Integrin signaling: cytoskeletal complexes, MAP kinase activation, and regulation of gene expression

Members of the integrin family of adhesion receptors mediate interactions of cells with the extracellular matrix. Besides their role in tissue morphogenesis by anchorage of cells to basement membranes and migration along extracellular matrix proteins, integrins are thought to play a key role in mediating the control of gene expression by the extracellular matrix. Studies over the past 10 years have shown that integrin-mediated cell adhesion can trigger signal transduction cascades involving translocation of proteins and protein tyrosine phosphorylation events. In this review, we discuss approaches used in our lab to study early events in integrin signalling as well as further downstream changes.     

Induction of a physical linkage between integrins and the cytoskeleton depends on intracellular calcium in an epithelial cell line

In most cases epithelial cells reveal a polarized distribution of integrin receptors in basolateral domains of the plasma membrane. To evaluate the functional state of integrin receptors in these restricted sites we were interested in the physical association of integrins with the cytoskeleton. Basically, we extracted cells with Triton X-100 to obtain detergent insoluble cytoskeleton fractions and used monoclonal antibodies for the detection of integrins linked to the cytoskeleton. We found that no permanent physical integrin-cytoskeleton associations exist in a confluent culture of the hepatocyte cell line mHepR1. However, incubation with anti-integrin antibodies and cross linking with a secondary antibody induced a physical linkage of beta1 as well as of different alpha subunits to the cytoskeleton. The association of integrins with the cytoskeleton was also inducible in suspended cells, which was detected in flow cytometric analyses and indicates that the formation of a physical integrin-cytoskeleton connection is independent of the localization of integrins, cell shape, and adhesion on a substrate. Using the Ca2+ chelators BAPTA-AM and EGTA, we found that intracellular calcium is a necessary prerequisite to induce a connection of integrins to the cytoskeleton. ATP or tauroursodeoxycholic acid, which provoke an intracellular calcium elevation, partly induced the formation of an integrin-cytoskeleton linkage. These results indicate the obvious role of intracellular calcium in integrin-dependent outside-in as well as inside-out signaling.     

Soluble ligands of the alpha v beta 3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells

Binding of substrate-bound extracellular matrix proteins to cell surface integrins results in a variety of cellular responses including adhesion, cytoskeletal reorganization, and gene expression. We have previously shown that addition of soluble SC5b-9, the complement-vitronectin complex, resulted in an RGD-dependent increase in lung venular hydraulic conductivity (Ishikawa, S., Tsukada, H., and Bhattacharya, J. (1993) J. Clin. Invest. 91, 103-109). To identify specific integrin(s) and signal transduction pathways that are responsive to soluble vitronectin-containing ligands, we exposed confluent bovine pulmonary artery cells to purified soluble human mono- or multimeric vitronectin, or SC5b-9, and determined the extent of endothelial cell protein tyrosine phosphorylation. Monomeric vitronectin (Vn) did not induce enhanced protein tyrosine phosphorylation. However, multimeric Vn and SC5b-9 elicited time- and concentration-dependent increases in tyrosine phosphorylation of numerous proteins. Antiserum against vitronectin, RGD peptides, and monoclonal and polyclonal antibodies against the alpha v beta 3 integrin blocked the vitronectin- or SC5b-9-induced enhanced accumulation of tyrosine phosphoproteins, while antibodies against beta 1 integrins and the alpha v beta 5 integrin did not. Clustering of the alpha v beta 3 integrin using monoclonal antibody LM609 caused a pattern of enhanced tyrosine phosphorylation similar to that caused by multimeric Vn and SC5b-9, suggesting that aggregation of alpha v beta 3 was critical for signaling. Among the proteins that underwent enhanced tyrosine phosphorylation in response to vitronectin were the cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing protein Shc, and p125FAK. We conclude that ligation of the alpha v beta 3 integrin by soluble ligands promotes enhanced phosphorylation of several proteins implicated in tyrosine kinase signaling and suggest that this pathway may be important in inflammatory states which are accompanied by accumulation of SC5b-9.     

A 16-element phased-array head coil

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.     

MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of beta 1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 micrograms/mL) and flow (shear stress, 12 dynes/cm2) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the beta 1 affinity-sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a beta 1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that "costimulatory" events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.     

T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.     

Characterization of the novel focal adhesion kinase RAFTK in hematopoietic cells

Protein tyrosine kinases (PTKs) mediate signals that respond to many pivotal cellular functions. Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and PTKs, is a critical control mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion kinase (FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signaling and plays a role in signal transduction pathways mediating cell adhesion, motility and anchorage-independent growth. Recently, we and others have identified a novel protein tyrosine kinase termed RAFTK, (also known as Pyk2 or Cak-beta), which is related to FAK. This review describes the role of RAFTK in various signaling cascades mainly in reference to hematopoietic cell lineages.     

Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells

Kaposi's sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130(cas). Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Cellular uptake properties of a 2''-amino/2''-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA

PURPOSE: Rhabdomyosarcomas (RMS) are heterogeneous in their clinical presentation, histology, and cytogenetics. The growth of some RMS cells has been found to be regulated by the tyrosine kinase insulin-like growth factor (IGF) type I receptor. However, RMS cells exhibit variable sensitivity to inhibitors of tyrosine kinases and IGF receptors. Collectively, these heterogeneous features suggest that differences exist in the growth regulatory pathways of RMS. The objective of this study is to identify active tyrosine kinase signal transduction pathways in embryonal and alveolar RMS cells. METHODS: RMS tumor samples and cell lines representing both embryonal and alveolar histologic subtypes have been analyzed by immunoprecipitation and immunoblotting techniques to characterize phosphotyrosyl protein patterns and to identify tyrosine phosphorylated proteins. RESULTS: RMS cells can be characterized based on the patterns of phosphotyrosyl proteins, including the phosphorylation status of the catenin-like protein Cas1 and the signal adapter protein SHC, and the activation of IGF type I receptor signaling cascades including the formation of SHC-GRB2 signal protein complexes and MAP kinase activation. CONCLUSIONS: Rhabdomyosarcomas, especially the embryonal histologic subtype, are heterogeneous at the level of tyrosine kinase signal transduction. It will be important to characterize the growth regulatory pathways active in individual RMS tumors before targeting molecular therapies to this malignancy.     

Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes beta(I) and delta

T cells activated via integrin receptors can polarize and start crawling locomotion with repeated cycles of cytoskeletal reassembly processes, many of which depend on phosphorylation. We demonstrate that protein kinase C (PKC) activation represents an essential event in induction of active T cell motility. We find that in crawling T cells triggered via cross-linking of integrin LFA-1 two PKC isoenzymes, beta(I) and delta, are targeted to the cytoskeleton with specific localization corresponding to the microtubule-organizing center (MTOC) and microtubules, as detected by immunocytochemistry and immunoblotting. Clustering of LFA-1 associated with its signaling function also occurs at the membrane sites adjacent to the MTOC. We further show that cells of a PKC-beta-deficient clone derived from parental PKC-beta-expressing T cell line can neither crawl nor develop a polarized microtubule array upon integrin cross-linking. However, their adhesion and formation of actin-based pseudopodia remain unaffected. Our data demonstrate the critical importance of the microtubule cytoskeleton in T cell locomotion and suggest a novel microtubule-directed intracellular signaling pathway mediated by integrins and involving two distinctive PKC isoforms.     

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.