A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

Abstract

The common model for integrin mediated signaling is based on integrin clustering and the potential for that clustering to recruit signaling molecules including FAK and src. The clustering model for transmembrane signaling originated with the analysis of the EGF receptor signaling and remains the predominant model. The roles for substrate-bound ligand and ligand occupancy in integrin-mediated signaling are less clear. A kinetic model was established using HT1080 cells in which there was a linear relationship between the strength of adhesion, the proportion of ␣5␤1 integrin that could be chemically cross-linked, and the number of receptor-ligand bonds. This graded signal produced a similarly graded response measured by the level of specific phosphorylation of FAK Y397. FAK Y397 phosphorylation could also be induced by antibody bound to the substrate. In contrast, clustering of ␣5␤1 on suspended cells with either antibody to ␤1 or by clustering of soluble ligand bound to ␣5␤1 induced the phosphorylation of FAK Y861 but not Y397. There were no differences in signaling when activating antibodies were compared with blocking antibodies, presence or absence of ligand. Only tethering of ␣5␤1 to the substrate was required for induction of FAK Y397 phosphorylation. INTRODUCTIONThere are several classes of transmembrane receptors that are involved in the transmission of signals from the extracellular space across the plasma membrane. It is a logical requirement of these systems that ligand binding to the extracellular domain results in a change in the cytoplasmic domain. How the signal is transferred from the extracellular domain is basic to the generation of the intracellular downstream signals. Allosteric proteins have been described in which the occupation of a binding site on one side of the protein can result in a conformational change that affects other binding sites. The interposition of a lipid bilayer between the receiving and effector domains of the transmembrane receptors poses limitations in the application of the allosteric model. Although a few of the receptor tyrosine kinase receptor systems have been analyzed in detail, the mechanism for transmembrane signal transduction by other receptor classes is less well understood.The EGF receptor is the best understood model. Receptor dimerization is initiated by the binding of EGF to extracellular domain 1 altering the conformation of the extracellular domain to generate a dimerization of receptors mediated through domain 2. This dimerization brings the cytoplasmic domains of two EGF receptors into proximity and allows the cross-phosphorylation of cytoplasmic domains by the encoded tyrosine kinase (Schlessinger, 2000). The generation of signals through receptor dimerization is a general theme. Among the tyrosine kinase receptors the mechanisms of generating the dimer vary from the use of bivalent ligands for growth hormone and erythropoietin (Kossiakoff and de Vos, 1998;Jiang and Hunter, 1999), to dimeric ligands for PDGF and VEGF (Wiesmann et al., 1997), to complexe...

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

“…In contrast, the Col I-induced FAK activation is tension-independent because the ligand GFOGER motif in Col I is readily accessible to integrin α2. Therefore, our data showed that integrin α subunits play crucial roles in determining the mechanosensitivity of FAK activation, as the adhesion of HT1080 cells to FN or Col I is dependent on integrin α5β1 (15,28) or α2β1 (29-31), respectively (SI Appendix, Fig. S2 and Fig.…”

“…Therefore, the ligation of α subunit and the ECM proteins converges at the integrin β1 activation and shares the mechanism of FAK activation. The clustering of integrin β1 by activating antibody AG89 was previously shown to induce the FAK phosphorylation on Tyr861, but not Tyr397, in the suspended HT1080 cells (28). 12G10 recognizes the βA domain of integrin β1 at the ligand-binding region (33), whereas AG89 recognizes the membrane proximal stalk-like regions of integrin β1 (34).…”

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix

“…MYO1E SH3-mediated autoinhibition of MYO1E motor function (26) may be released by MYO1E binding to FAK. MYO1E binding to FAK then tethers FAK to actin, which may promote endocytosis (18) and could explain why FAK function is tightly linked to actomyosin dynamics and integrin endocytosis (10,27). Upon FAK Y397 autophosphorylation, SRC binds FAK through SH2 domain interaction, which, in turn, releases SRC SH3 to interact with FAK PRR1, thereby possibly replacing MYO1E.…”

“…Y397 is nonphosphorylated (9). Upon activation by tethering (10) or other stimuli that induce conformational change (11), the linker region is exposed and Y397 becomes autophosphorylated, leading to the recruitment of the protooncogene SRC. FAK and SRC then form a transient complex, which stabilizes FAK in its active conformation and induces changes in cell shape and focal adhesion turnover in vitro (12).…”

$$\upalpha 5\upbeta $$ α 5 β 1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

“…It has been previously shown that the strength of HT1080 cell adhesion to either FN coatings or fibrillar FN matrices is drastically decreased upon antibody blocking of α 5 β 1 -integrin [37,38]. In contrast, blocking of α v β 3 -integrin does not significantly affect fibrosarcoma cell adhesion [38,39], although this integrin type binds to fibrillar FN [40] and is important for adhesion to FN in other cell lines. Upon α 5 β 1 -integrin blocking, the reduced migration speed on FN coatings suggests that cell binding via α 5 β 1 -integrin is necessary for promoting migration in 2D.…”