Focal adhesion kinase-like
Hepatocyte growth factor (HGF) stimulates a significant increase in the tyrosine phosphorylation of FAK in human embryonic kidney 293
cells. This stimulation is independent of cell adhesion or the integrity of the actin cytoskeleton, suggesting potentially different mechanisms by which the
HGF receptors and integrins regulate the tyrosine phosphorylation of FAK. These results also suggest that the activation of Src upon HGF stimulation is
likely to be one, if not the only, of the mechanisms responsible for the HGF-induced tyrosine phosphorylation of FAK. A
mutation in the Grb2 binding site Tyr-925 of FAK partially abolishes its increase in HGF-induced phosphorylation. HGF
stimulates the association of FAK with Grb2 in vitro and in intact cells and evidence is provided that FAK might contribute to the activation of
mitogen-activated protein kinase through Ras in HGF signaling by functioning as an adapter molecule (Chen, 1998).
The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting
the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Evidence has been found that integrins cause a significant and protracted activation of Jun
NH2-terminal kinase (JNK: Drosophila homolog Basket), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK requires the association of focal adhesion kinase (FAK) with a Src kinase and p130CAS, the phosphorylation of p130CAS, and subsequently, the recruitment of Crk. Ras and PI-3K
are not required. FAK-JNK signaling is necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling
that is consistent with the role of Jun in both proliferation and transformation (Oktay, 1999).
What is the mechanism by which FAK activates JNK? Upon activation, FAK undergoes autophosphorylation at tyrosine 397 and combines with the SH2 domain of Src or
Fyn. The most prominent substrates of the FAK/Src complex are the docking adaptor proteins p130CAS and paxillin. Both contain tyrosine phosphorylation sites conforming to the consensus for binding to the adaptor protein Crk. However, while paxillin has only two such sites and does not appear to associate efficiently with Crk in response to integrin ligation, p130CAS contains nine Crk-binding motifs and associates well with Crk in cells adhering to fibronectin. The expression of dominant-negative versions of FAK, Src, p130CAS, and Crk suppress the activation of JNK by integrins. These findings provide evidence that integrin-mediated activation of JNK requires the association of FAK with Src (or Fyn) and p130CAS, and the recruitment of Crk. Thus, it appears that the beta1 and alphav integrins activate JNK and ERK via two separate pathways. By contrast, the alpha6beta4 integrin, which is presumably unable to activate FAK because it does not contain the sequences required for its recruitment, is coupled to JNK signaling via the Ras-PI-3K-Rac pathway. The identity of genes regulated by JNK is largely unknown, but they must include genes important for cell proliferation. The evidence for this is several fold: (1) deregulated expression of c-Jun or its mutated viral version v-Jun is sufficient to cause neoplastic transformation of primary avian and mammalian fibroblasts; (2) primary fibroblasts derived from c-Jun minus mice display a severe proliferation defect; and (3) several oncoproteins, including v-Src, activated Ras, v-Crk, Bcr-Abl, and Met, potently activate JNK and there is evidence to suggest that this activation is required to cause neoplastic transformation. Despite the clear requirement for c-Jun transcriptional activity in cell proliferation, it has been difficult to identify a physiological, nonstress stimulus for JNK consistent with its role in the regulation of AP-1 transcription. With the notable exception of EGF, mitogenic neuropeptides, and muscarinic receptor ligands, which indeed activate FAK or the related kinase PYK-2, most growth factors cause a relatively modest activation of JNK. The results indicating that integrin ligation causes a significant activation of JNK and TRE-dependent transcription provide a physiological stimulus for JNK signaling that is consistent with its role in the control of cell proliferation (Oktay, 1999 and references).
Most transformed cells have lost anchorage and serum dependence for growth and survival. When serum
is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts
and endothelial cells. This study sought to identify survival sequences in FAK and
signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. Binding of the
SH3 domain of p130Cas (see CAS/CSE1 segregation protein) to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is
withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4)
pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but JNK does not colocalize with FAK in
fibroblasts cultured on collagen. which does not support survival. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is
absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented here. Thus, survival signals from extracellular matrix
and serum are transduced by FAK via two distinct pathways (Almeida, 2000).
Whether FAK tyrosine kinase
mediates the activation of mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. The activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects are phosphatidylinositol 3-kinase-independent, as FAK effectively stimulates Akt, and wortmannin suppresses Akt but not ERK or JNK activation. Activation of ERK correlates with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, evidence is provided that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent
activation of a biochemical route dependent on small GTP-binding proteins of the Rho family (Igishi, 1999).
The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP
kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell
growth. Expression of constructs that constitutively activate focal adhesion kinase (FAK) rescue the defect in serum activation of
MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocks both the rescue of suspended cells by the
activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK(-/)- mouse embryo fibroblasts is
adhesion-insensitive, and reexpression of FAK restores its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in
suspension, but expression of constructs that constitutively activate FAK enhance their anchorage-independent growth without increasing adherent
growth. V-src, which activates both Ras and FAK, induces MAP kinase activation that is insensitive to loss of adhesion, and is blocked by a
dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and
that bypassing this mechanism contributes to anchorage-independent growth in transformed cells (Renshaw, 1999).
The potential role and mechanisms of integrin signaling through FAK in cell cycle regulation has been analyzed by using tetracycline-regulated
expression of exogenous FAK and mutants. Overexpression of wild-type FAK accelerates G1 to S phase transition. Conversely,
overexpression of a dominant-negative FAK mutant DeltaC14 inhibits cell cycle progression at G1 phase and this inhibition requires the Y397 in
DeltaC14. Biochemical analyses indicate that FAK mutant DeltaC14 is mislocalized and functions as a dominant-negative mutant by competing with
endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion.
Consistent with this, inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKDeltaC14, is observed
in transient transfection assays using primary human foreskin fibroblasts. DeltaC14 blocks cyclin D1 upregulation and induces
p21 expression, while wild-type FAK increases cyclin D1 expression and decreases p21 expression. Taken together, these results have identified FAK
and its associated signaling pathways as a mediator of the cell cycle regulation by integrins (Zhao, 1998).
Focal adhesion kinase associates with integrin receptors, and FN-stimulated phosphorylation of
FAK at Tyr-397 and Tyr-925 promotes the binding of Src family protein tyrosine kinases (PTKs) and Grb2, respectively. To
investigate the mechanisms by which FAK, c-Src, and Grb2 function in Fibronectin-stimulated signaling events
to ERK2, wild type and mutant forms of FAK were expressed in human 293 epithelial cells by transient
transfection. FAK overexpression enhances FN-stimulated activation of ERK2 approximately 4-fold.
This is blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN
stimulation of ERK2 is Ras-dependent. FN-stimulated c-Src PTK activity is enhanced by wild
type FAK expression, whereas FN-stimulated activation of ERK2 is blocked by expression of the
c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of
FAK enhances activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK does not.
Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhances p130(Cas) association
with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced
Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects
of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential
for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the
formation of a FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2
mitogen-activated protein kinase activation (Schlaepfer, 1997).
G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment
of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have
been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex
assembly and receptor tyrosine kinase activation, the relative contribution of each to activation of the Erk 1/2 cascade following
stimulation of endogenous GPCRs in three different cell types has been determined. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which
depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic
acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced
mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive
to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but
has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to
both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells.
The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase,
Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by Src kinase inhibitors suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for
signals originating from either scaffold (Della Rocca, 1999).
The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important
role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38
MAPK signaling pathway are currently unknown. Focal adhesion kinase-related tyrosine kinase RAFTK (also known as
PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or
cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK
(RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway,
whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. The dominant-negative mutant of
MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells
with a membrane-permeable calcium chelator, inhibits
MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the
p38 MAPK signaling pathway in response to certain cytotoxic agents (Pandey, 1999).
Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. The transcription factor KLF8 has been identified as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant DeltaC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated directly by KLF8 binding to the GT box A and by an indirect mechanism, through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, these data provide further insight into molecular mechanisms for FAK to regulate cell cycle progression (Zhao, 2003).
FAK and its associated signaling pathways mediate cell cycle progression by integrins. The potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation was investigated by using tetracycline-regulated expression system as well as chimeric molecules. Induction of Pyk2 inhibits G(1) to S phase transition whereas comparable induction of FAK expression accelerates it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increases cell cycle progression as does
FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibit cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicate that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 have little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibit Erk activation in cell adhesion whereas FAK and PFhy1 stimulate it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. While FAK and PFhy1 are present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) are tyrosine phosphorylated and associated with Src and Fyn. This suggests that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant DeltaC14 by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition (Zhao, 2000).
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