Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase.

We have shown previously that cell adhesion or platelet-derived growth factor (PDGF) promotes the in vivo association of focal adhesion kinase (FAK) with phosphatidylinositol (PI) 3-kinase. In vitro experiments indicated that this interaction was mediated by the p85 subunit of PI 3-kinase and dependent on the tyrosine phosphorylation of FAK. Here we report data suggesting that the major autophosphorylation site of FAK (Tyr-397) is the binding site for the SH2 domains of p85 in vitro and is also required for the association of FAK with PI 3-kinase in vivo. We also show that Tyr-397 is responsible for the increased FAK:PI 3-kinase association upon PDGF stimulation, implying that no additional site of FAK was involved in its binding to PI 3-kinase after PDGF stimulation. Finally, we present evidence that the interaction of PI 3-kinase with Tyr-397 of FAK stimulates its activity. Together, these results suggest that FAK activation and autophosphorylation at Tyr-397 may lead to its association with PI 3-kinase through the SH2 domains of p85, which can subsequently activate PI 3-kinase during cell adhesion.

Interaction of focal adhesion kinase with cytoskeletal protein talin.

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 (Zachary, I., and Rozengurt, E. (1992) Cell 71, 891-894; Schaller, M., and Parsons, J. T. (1993) Trends Cell Biol. 3, 258-262). Here, we report data suggesting a possible association of FAK with the cytoskeletal protein talin in NIH 3T3 cells. We have identified a 48-amino acid sequence in the carboxyl-terminal domain of FAK necessary for talin binding in vitro. Furthermore, we have correlated the ability of integrin to induce FAK phosphorylation with its ability to bind talin 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.

Control of stable lamellipodia formation by expression of cell surface beta 1,4-galactosyltransferase cytoplasmic domains.

Mesenchymal cell migration on basal lamina is mediated, in part, by the binding of cell surface beta 1,4-galactosyltransferase (GalTase) to specific N-linked oligosaccharides in the E8 domain of laminin. On migrating cells, surface GalTase is anchored to the cytoskeleton; when GalTase is prevented from associating with the cytoskeleton, lamellipodia formation and subsequent migration are inhibited. To define better the involvement of GalTase-cytoskeleton interactions in cell motility, we examined the lamellipodia formation, polarity and migratory behavior of stably transfected 3T3 fibroblasts expressing increased or decreased levels of GalTase capable of interacting with the cytoskeleton. Initially, the motile behavior of individual cells was quantified in the absence of exogenous stimuli. Cells that overexpress GalTase binding sites for the cytoskeleton changed their polarity more frequently and translocated more erratically than did control cells when assayed on laminin substrata. These differences were not observed, however, when cells were plated on fibronectin, which does not contain binding sites for surface GalTase. GalTase-transfected cells were also assayed for their ability to polarize in response to a specific stimulus. In this case, the ability of a cell to reorient towards a gradient of platelet-derived growth factor was found to be directly proportional to the amount of GalTase associated with the cytoskeleton. Differences in response to platelet-derived growth factor were not due to differences in growth factor binding. Indirect immunofluorescence showed that altering the level of GalTase did not affect the ventrally distributed pool of GalTase stably associated with the cytoskeleton; however, stress fiber formation was inhibited. Thus, increasing surface GalTase binding sites for the cytoskeleton leads to erratic, multipolar behavior in the absence of any vectorial stimulus, but the ability to form a functional lamellipodium in response to a stimulus is dependent upon the amount of surface GalTase associated with the cytoskeleton. Apparently, cells are able to regulate cytoskeletal assembly and lamellipodial stability by altering the expression and/or affinity of appropriate matrix receptors, such as GalTase, and their corresponding binding sites in the cytoskeleton.

Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.

Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites.