Focal adhesion kinase-like
The interaction of cells with extracellular matrix proteins plays a
critical role in a variety of biological processes. Recent studies suggest
that cell-matrix interactions mediated by integrins can transduce
biochemical signals to the cell interior that regulate cell proliferation
and differentiation. These studies have placed the focal adhesion kinase
(FAK), an intracellular protein tyrosine kinase, in a central position in
integrin-initiated signal transduction pathways. An association of FAK with the cytoskeletal protein talin occurs in NIH 3T3
cells. A 48-amino acid sequence in the carboxyl-terminal
domain of FAK necessary for talin binding in vitro has been identified. The ability of integrin to induce FAK phosphorylation with its
ability to bind talin has been demonstrated using a mutant integrin lacking the carboxyl-terminal
13 amino acids. These studies suggest talin may be a mediator for FAK
activation in signaling initiated by integrins and may provide an
explanation for the dependence on the integrity of actin-cytoskeleton of
multiple intracellular signaling pathways converging to FAK activation and
autophosphorylation (Chen, 1995).
Insulin receptor substrate-1 (IRS-1: Drosophila homolog Chico) is a major substrate of insulin and insulin-like growth factor-I receptors, which upon phosphorylation on tyrosine
docks several signaling molecules. Recently, IRS-1 was found to interact with alphav beta3 integrins upon insulin stimulation. Integrins are transmembrane
proteins that play an important role in adhesion between cells and between cells and extracellular matrix. One of the major proteins implicated in integrin
signaling is pp125(FAK), a cytosolic tyrosine kinase, which upon integrin engagement becomes tyrosine-phosphorylated and subsequently binds to c-Src.
A mammalian two-hybrid system was established to show that pp125(FAK) binds to IRS-1. This association depends largely on the C terminus of
pp125(FAK) but not on pp125(FAK) tyrosine kinase activity. Furthermore, co-immunoprecipitation of pp125(FAK) with IRS-1 was observed in 293
cells, suggesting a possible biological function of this association. When IRS-1 is expressed in 293 cells together with pp125(FAK) or Src,
extensive IRS-1 tyrosine phosphorylation is found. In pp125(FAK)-expressing cells, this is concomitant with increased association of IRS-1 with Src homology
2-containing proteins such as growth factor receptor-bound protein 2, phosphatidylinositol (PI) 3-kinase p85alpha subunit, and Src homology 2-containing
protein-tyrosine phosphatase-2. In addition, pp125(FAK)-induced association of IRS-1 with PI 3-kinase resulted in increases PI 3-kinase activity. In
contrast, no change in mitogen-activated protein kinase activity is observed, indicating that pp125(FAK)-induced association between IRS-1 and
growth factor receptor-bound protein 2 does not affect the mitogen-activated protein kinase pathway. Engagement of integrins
induces IRS-1 tyrosine phosphorylation. Considering these results together, it is suggested that integrins and insulin/insulin-like growth factor-I receptor
signaling pathways converge at an early point in the signaling cascade, which is the IRS-1 protein (Lebrun, 1998).
Pyk2 is a cytoplasmic tyrosine kinase that is related to
focal adhesion kinase (FAK) and can be activated by a variety of stimuli
that elevate intracellular calcium. Pyk2 and
FAK tyrosine phosphorylation are regulated differentially by
integrin-mediated cell adhesion and soluble factors both in rat aortic
smooth muscle cells, which express endogenous Pyk2 and FAK, and in
transfected Chinese hamster ovary cells. Pyk2 is
diffusely present throughout the cytoplasm, while FAK is localized in focal
contacts as expected, suggesting that the different localization may
account for their differential regulation. By analyzing a chimeric protein
contain
N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, evidence is provided that the distinctive C-terminal domains of Pyk2 and
FAK are responsible for their differential regulation by integrins and
soluble stimuli as well as their subcellular localization. FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, are correlated
with their regulation by integrins and focal contact localization. These
results
demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer
their differential regulation by different subcellular localization and
association with the cytoskeletal protein talin (Zheng, 1998).
The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, expression cloning was used to identify cDNAs that encode potential FAK-binding proteins. A new member of the GTPase-activating protein (GAP) family of GTPase regulators is described. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. It is suggested that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events (Hildebrand, 1996).
Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular
responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been
impeded by extensive redundancy between family members. Mouse embryos were generated
harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple
mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular
mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and
tested for their ability to respond to growth factors or plating on extracellular matrix. While Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced
signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix
proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal
adhesion kinase FAK, is nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent
with previous reports demonstrating the importance of FAK for cell migration, SYF cells display
reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis
and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling
by extracellular matrix-coupled receptors (Klinghoffer, 1999).
Low density lipoprotein (LDL) is known to sensitize platelets to agonists via integrin mediated outside-in signaling. As outside in signaling is
associated with phosphorylation of p125(FAK), the effect of LDL on p125(FAK) phosphorylation in platelets was investigated. LDL induces
p125(FAK) phosphorylation in a dose- and time- dependent manner. The phosphorylation is independent of ligand binding to integrin alphaIIbbeta3 and
aggregation, such in contrast to alpha-thrombin-induced p125(FAK) phosphorylation, that critically depended on platelet aggregation. Platelets from
patients with Glanzmann's thrombastenia show the same LDL- induced phosphorylation of p125(FAK) as control platelets, whereas alpha-thrombin
completely fails to phosphorylate the kinase in the patients platelets. LDL signaling to p125(FAK) is independent of integrin alpha2 beta1, the
FcgammaRII receptor, and the lysophosphatidic acid receptor and not affected by inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or
p38(MAPK). Phosphorylation of p125(FAK) by LDL is strongly inhibited by cyclic AMP. These observations indicate that LDL is a unique platelet
agonist, as it phosphorylates p125(FAK) in platelet suspensions, under unstirred conditions and independent of integrin alphaIIb beta3 (Hackeng, 1999).
Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and
Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An
increase in complex formation between FAK and Src in response to bombesin can be detected within 1 min, reaches a maximum after 10 min, and
declines toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex
formation. Bombesin stimulates FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity
of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibits both
FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations also induces FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase.
Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached
cells through different signal transduction pathways (Salazar, 1999).
Addition of insulin growth factor-I (IGF-I) to quiescent Swiss 3T3 cells rapidly induces tyrosine phosphorylation of the p130Crk-associated substrate
(p130Cas; see CAS/CSE1 segregation protein), a novel adaptor protein localized at focal adhesions. Half-maximal effect is obtained at 0. 6 nM. IGF-I also promotes the formation of a
complex between p130(Cas) and c-Crk and elicits a parallel increase in the tyrosine phosphorylation of p125(Fak) and paxillin. IGF-I-induced
p130(Cas), p125(Fak), and paxillin tyrosine phosphorylation can be dissociated from mitogen-activated protein kinase kinase, p70(S6K: see Drosophila RPS6-p70-protein kinase), and protein
kinase C activation. In contrast, the structurally unrelated phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 markedly attenuate the
increase in tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin induced by IGF-I. Cytochalasin D, which disrupts the network of actin
microfilaments, completely prevents tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin and the formation of a p130(Cas)-Crk complex in
response to IGF-I. Thus, these results identify a phosphatidylinositol 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton to
induce tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin in response to IGF-I and suggest that tyrosine phosphorylation of these focal
adhesion proteins, together with the recruitment of c-Crk into a complex with p130(Cas), may play a novel role in IGF-I signal transduction (Casamassima, 1998).
HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is
phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G
protein-coupled receptors has not been reported. HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit
C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increases in a time- and
dose-dependent manner. Dibutyryl cAMP and forskolin have little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 fails to
detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin,
which selectively blocks G(i/o) signaling, also has no effect. Increasing cytosolic Ca(2+) with ionomycin stimulates HEF1 phosphorylation and preventing
any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocks the calcitonin-induced
tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induces HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor
calphostin C completely inhibits both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induces the
tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D,
which disrupts actin microfilaments, prevents the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine
phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton (Z. Zhang, 1999).
Tyrosine phosphorylation of FAK and paxillin are decreased during dibutyryl cyclic AMP-induced (dB-cAMP) process formation in
astrocytes. When astrocytes in suspension are treated with dB-cAMP, no alteration in morphology or tyrosine phosphorylation is observed, suggesting that
both phenomena are linked and adhesion dependent. Furthermore, genistein, a tyrosine kinase inhibitor, can induce process formation in such cells,
underscoring the significance of protein tyrosine kinases in maintaining the morphology of adherent cells. Finally, endothelin-1, a vasopeptide which is
known to inhibit process formation in astrocytes, inhibits the tyrosine dephosphorylation of proteins associated with dB-cAMP treatment. These results
suggest that the formation of asymmetric processes in astrocytes results from a coordinated set of alterations in the actin cytoskeleton as well as the
adhesion of the cell to the substratum. Modification of the properties of such molecules is required for process formation and the dynamic modulation of
astrocytic morphology in vitro and in vivo (Padmanabhan, 1999).
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. 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 is 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 induces the activation
of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reaches a maximum after 45
min of stimulation; in contrast, PYK-2 activation peaks at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2
redistribute from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation
is blocked when lymphoblasts are pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. These 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 (Rodriguez-Fernandez, 1999).
ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, ASAP1 has been identified as a major binding partner of FAK. GST pull-down and coIP assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. It is concluded from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK (Liu, 2002).
Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. This study reports that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, these results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity (Zhai, 2003).
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