An Examination of Focal Adhesion Formation and Tyrosine Phosphorylation in Fibroblasts Isolated from src¯, fyn¯, and yes¯ Mice

Abstract

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125^FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59^fyn, pp60^v-src, and activated pp60^c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60^c-src p59^fyn, or pp62^c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60^src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59^fyn and pp62^c-yes, and particularly in those lacking pp60^c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.

Key Words:

• focal adhesion kinase
• fibronectin
• cytoskeleton