Tyrosine phosphorylation of the focal adhesion kinase pp125^FAK during development: relation to paxillin

Significant changes in the level of protein tyrosine phosphorylation accompany avian embryonic development. A comparison of different tissues reveals that a similar and remarkably restricted complement of proteins is modified in this manner. In each case the major proteins detected using anti-phosphotyrosine antibodies have molecular masses of approximately 170, 150, 125, 70 and 50 kDa. As a first step in determining the function of this protein modification in embryogenesis we have initiated a study to identify these phosphoproteins. We have previously reported that the 70 kDa band is paxillin, a component of actin-membrane attachment sites associated with regions of cell adhesion (Turner, C.E. (1991) J. Cell Biol. 115, 201-207). We report here that the 125 kDa phosphotyrosine-containing protein is the tyrosine kinase pp125^FAK, a protein that co-localizes with paxillin at sites of adhesion (Schaller et al. (1992) Proc. Nat. Acad. Sci. USA 89, 5192-5196). Tyrosine phosphorylation of both pp125^FAK and paxillin was detected at low levels as early as embryonic day 3 and increased steadily during the first half of development, reached a maximum between embryonic days eight and twelve, and declined to background levels prior to hatching. Paxillin protein expression also increased during the first half of embryogenesis, suggesting little change in the overall phosphorylation of this protein through embryonic day 8. In contrast, pp125^FAK, following an initial increase, is expressed at a constant high level during these early embryonic stages, implying an increase in its overall phosphotyrosine content. In the second half of embryonic development pp125^FAK expression decreased in parallel with the decrease in tyrosine phosphorylation of this and the other phosphopeptides. The alternatively-spliced 41/43 kDa form of pp125^FAK is also present in the embryo, but at much reduced levels, and is not phosphorylated on tyrosine. At the stage of development corresponding to the highest level of phosphorylation of pp125^FAK (day 12) approximately 54% of the pp125^FAK was phosphorylated on tyrosine. pp125^FAK and paxillin exhibit a similar tissue distribution with the exception of brain where only pp125^FAK was detected. Immunoprecipitates of pp125^FAK from embryonic smooth muscle extract exhibited tyrosine kinase activity that phosphorylated pp125^FAK and a 60 kDa protein. This immune complex also catalyzed the tyrosine phosphorylation of purified paxillin. Our identification of intracellular proteins associated with cell attachment, namely pp125^FAK and paxillin, as two of the major targets for tyrosine phosphorylation during embryogenesis suggests that this protein modification may contribute to the regulation of important cell adhesion events during embryonic morphogenesis.