Targeting Membrane-localized Focal Adhesion Kinase to Focal Adhesions

Abstract

In the present study, we examined regulation of activated focal adhesion kinase localization in focal adhesions. By using focal adhesion kinase fused to an inert transmembrane anchor, we found that the focal contact targeting region within focal adhesion kinase was preserved in the membrane-targeted fusion protein. However, upon tyrosine phosphorylation, full-length focal adhesion kinase became excluded from focal adhesions. This negative regulation of localization could be abolished by mutating key amino acid residues of focal adhesion kinase shown previously to be involved in adhesion-mediated signal transduction. Hyper-phosphorylation of endogenous focal adhesion kinase induced by pervanadate resulted in a similar reduction of localization at focal adhesions. We also show here that Src family kinases are essential for the phosphorylation-dependent exclusion of focal adhesion kinase from focal adhesions. We propose here a molecular model for the tyrosine phosphorylation-dependent regulation of focal adhesion kinase organization involving Src kinases and an inhibitory phosphorylation of the C-terminal (Tyr-925) tyrosine residue.Adhesion-mediated signaling triggers tyrosine phosphorylation of a multitude of proteins, including paxillin, tensin, and focal adhesion kinase (FAK) 1 (1-4). FAK phosphorylation following adhesion depends on its association with Src family kinases, leading to the formation of multimolecular signaling complexes in which FAK serves as a scaffold (5-7). Following activation, residue Tyr-397 of FAK becomes auto-phosphorylated, resulting in the formation of an SH2-docking site (5). Src family kinases then associate with Tyr-397 and phosphorylate several other tyrosine residues in FAK, including Tyr-925 that resides within the focal adhesion targeting (FAT) sequence at the FAK C terminus (5). The adaptor protein Grb2 then binds phosphorylated Tyr-925 and forms a signaling complex that includes the nucleotide exchange factor Sos and the small GTP-binding protein Ras (5). This sequence of events contributes to activation of the ERK1/2 response (5). Two additional pathways lead to FAK-mediated activation of ERK1/2: one involves binding of the adaptor protein p130 Cas to the prolinerich region of FAK via an SH3 domain interaction, and the second is the result of FAK association with the adaptor protein Shc (8 -10).The mechanisms that underlie FAK activation are not yet clear. Although FAK lacks any myristoylation or farnesylation signals, membrane translocation of FAK could be initiated by direct association with the ␤ 1 integrin tail (11). Close indirect interaction between FAK and integrin cytoplasmic tails may also be mediated via talin or paxillin (12, 13). Indeed, several studies (11,14) have demonstrated that a membrane-translocated form of FAK (following fusion to an inert transmembrane anchor) results in an adhesion-independent, constitutive phosphorylation of the molecule. This phosphorylated state of FAK depends on its association with Src and is accompanied by constitutive association ...

Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407

“…These findings seem to indicate that FAK Tyr 407 phosphorylation might inhibit the access of additional phosphates on Tyr 397 , perhaps because of increased negative charge repulsion. It has been known that FAK molecules phosphorylated at Tyr 397 reside close to the plasma membrane (21,22), and the absence of Tyr 397 phosphorylation decreases its residency at focal adhesions but not in cytosol (22). Consistent with this notion, higher levels of Tyr 407 phosphorylation were observed in cytosolic FAK versus membrane-bound FAK (Fig.…”

The interplay between Src and integrins in normal and tumor biology

“…In contrast, CHO cells expressing Y925F FAK mutant were found to migrate at a similar rate to wild-type FAK (Cary et al, 1998). Phosphorylation of this residue may also regulate the localization of FAK to focal adhesions (Katz et al, 2003).…”

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

“…While tyrosine phosphorylation of GIT1 residues within the FAH domain does not appear to be an important contributor to binding, as it is in FAK [33], tyrosine phosphorylation elsewhere in GIT proteins does appear important. First, we identified increased binding of paxillin to the Y563F mutant ( Figure 7).…”

“…Focal adhesion kinase binding to paxillin through its four-helix bundle FAT domain, and therefore localization to focal adhesions, is regulated in part through phosphorylation of FAK on tyrosine 925, a residue in helix 1 of the FAT domain itself [33]. When phosphorylated at tyrosine 925 by Src family members, FAK is released from focal complexes [33]. We sought to determine whether GIT1 association with paxillin might also be regulated through tyrosine phosphorylation.…”

“…One reason for this difference may be that we have analyzed this interaction with isolated proteins, whereas in living cells other regulatory modes might compensate for changes due to aspartate mutation of the phosphorylated serines, or the aspartate mutation itself might not truly mimic a phosphate in this instance. Nevertheless, phosphorylation of paxillin within the LD4 motif clearly does play a role in regulating GIT protein binding to paxillin, and bears further investigation.While tyrosine phosphorylation of GIT1 residues within the FAH domain does not appear to be an important contributor to binding, as it is in FAK [33], tyrosine phosphorylation elsewhere in GIT proteins does appear important. First, we identified increased binding of paxillin to the Y563F mutant ( Figure 7).…”