Protein kinase C-dependent tyrosine phosphorylation of p130cas in differentiating neuroblastoma cells

The cell signaling docking protein p130(cas) became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60(c-src). The TPA-induced p130(cas) phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130(cas) phosphorylation was biphasic, However, the increase in tyrosine phosphorylation of p130(cas) was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-epsilon induced p130(cas) tyrosine phosphorylation, pp60(c-src), known to directly phosphorylate p130(cas) in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130(cas) phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130(cas), Also, overexpression of src induced phosphorylation of p130(cas). p130(cas) protein and phosphorylated p130(cas) were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130(cas) phosphorylation decreased transiently (within minutes), Growth cones isolated from these cells were virtually devoid of phosphorylated p130(cas). These data suggest a function for p130(cas) as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.