The ability of
mitogens to rapidly induce
tyrosine phosphorylation of cellular
proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major
protein kinase C (PKC) substrate with
protein scaffolding and
tumor suppressive properties, becomes
tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with
epidermal growth factor (
EGF),
platelet-derived growth factor (PDGF), or
fetal calf serum in the presence of
pervanadate, but not by treatment with
insulin or
insulin-like growth factor-1. The relative
phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures.
Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl
tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src
enzyme failed to directly phosphorylate SSeCKS in vitro.
EGF failed to induce SSeCKS
tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the
EGF receptor is probably not the direct
kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to
fibronectin failed to significantly induce SSeCKS
tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a
growth factor receptor-FAK rather than an
integrin-FAK pathway. Moreover, PDGF could induce SSeCKS
tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than
kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along
F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with
biotin-
phalloidin-bound
F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with
F-actin in ultracentrifugation assays. These data suggest that
mitogen-induced, FAK-dependent
tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in
mitogen-induced cytoskeletal reorganization.