Protein Kinase C Regulates v [ 35-dependent Cytoskeletal Associations and Focal Adhesion Kinase Phosphorylation

Integrins av[33 and av[35 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin av133 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas av135 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of 133 or 135 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that av135 induced colocalization of talin, ot-actinin, tensin, and actin very weakly relative to ~v[33. eLv135 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to av[35-expressing cells induced spreading, increased colocalization of tx-actinin, tensin, vinculin, p130 cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, a-actinin, tensin, and actin for both av133 and txv135. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by etvl~5. Our resuits also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin. I NTEGRINS mediate adhesion of cells to extracellular matrix proteins and to other cells. During adhesion, they also form linkages to the cytoskeleton and regulate intracellular signaling pathways, thereby coordinating attachment to cell architecture and gene expression (Burridge et al., 1988; Hynes, 1992; Miyamoto et al., 1995; Schwartz et al., 1995). Despite significant progress towards understanding these phenomena, much remains to be learned about how integrin-dependent events control cell functions. Integrins are heterodimers, and binding specificity is generated by varied combinations of the 14 ~x and 8 13 subunits identified to date (Hynes, 1992). Binding specificities show a high degree of redundancy, however, such that both integrins and their ligands bind multiple partners. Evidence is emerging that individual receptors mediate distinct functions and trigger distinct signaling pathways (Hynes, 1992; Sastry and Horwitz, 1993; Schwartz et al., 1995). After initial ligand binding, integrins recruit a number of signaling and cytoskeletal proteins to sites of adhesion (Burridge et al., 1988; Miyamoto et al., 1995). While the precise organization of these structures remains to be determined, ~x-actinin and talin have been shown to bind directly to sequences in the 131 cytoplasmic domain (Horwitz Address all correspondence to M.A. Schwartz, Department of Vascular Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-7140. Fax: (619) 784-7360. et al., 1986; Otey et al., 1990). Similar sequences are present in several other integrin 13 chains, suggesting that these interactions are conserved. Both c~-actinin and talin have been reported to bind vinculin (Belkin and Koteliansky, 1987; Wachsstock et al., 1987), which can associate with both tensin and paxillin (Wilkins et al., 1986; Turner et al., 1990). Focal adhesion kinase (FAK) 1 has been reported to bind to peptides from the integrin 131 cytoplasmic domain, to talin, to paxillin, and to p130 cas (Polte and Hanks, 1995; Bellis et al., 1995; Chen et al., 1995; Schaller and Parsons, 1995; Schaller et al., 1995). There is evidence that these linkages are subject to regulation, although the details are not well understood. Integrin function is regulated by cytokines and other soluble factors in a variety of systems. In most cases, these factors modulate the avidity of integrins for their ligands, by altering either the conformation of the receptor or the lateral distribution in the plasma membrane (for review see Schwartz et al., 1995). Integrin av135 represents an interesting exception to this rule, since it binds very well to its ligand vitronectin (VN), but in the absence of exogenous soluble factors, it fails to promote cell spreading or migration (Wayner et al., 1991; Klemke et al., 1994) 2. By 1. Abbreviations used in this paper: FAK, focal adhesion kinase; FN, fibronectin; PKC, protein kinase C; VN, vitronectin. 2. Brooks, P.C. , R.L. Klemke, S. Sch6n, J.M. Lewis, M.A. Schwartz, and D.A. Cheresh. Manuscript submitted for publication. © The Rockefeller University Press, 0021-9525/96/09/1323/10 $2.00 The Journal of Cell Biology, Volume 134, Number 5, September 1996 1323-1332 1323 on July 6, 2017 jcb.rress. org D ow nladed fom contrast, the 133 subunit, which is 56% homologous to 135, also pairs with av and binds VN but induces spreading and migration in the same cells in the absence of cytokines (Wayner et al., 1991; Filardo et al., 1995). However, upon stimulation with either phorbol ester or cytokines to activate protein kinase C (PKC), av135 acquires the ability to mediate cell spreading and migration in vitro (Klemke et al., 1994; Yebra et al., 1995). This PKC-dependent activation correlates with the ability of av135 to promote anglogenesis by endothelial cells and metastasis by tumor cells in vivo (Friedlander et al., 1995). 2 The aim of this study was to investigate how PKC regulates the function of av135. While previous studies suggested that PKC may alter connections to the cytoskeleton, 2 the molecular mechanism explaining this observation remains unclear. We therefore assayed the ability of c~vl35 to induce localization of a number of cytoskeletal and signaling proteins before and after activation of PKC. Our resuits show that PKC does regulate linkages to specific cytoskeletal proteins. We also find that FAK colocalized with etv135 independent of PKC but that its phosphorylation is PKC dependent. Materials and Methods