PROTEIN-KINASE C-DEPENDENT TYROSINE PHOSPHORYLATION OF P130(CAS) IN DIFFERENTIATING NEUROBLASTOMA-CELLS

The cell signaling docking protein p130(cas) became tyrosine-phosphory lated in SH-SY5Y human neuroblastoma cells during induced differentiat ion with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a com bination of basic fibroblast growth factor (bFGF) and insulin-like gro wth factor-I (IGF-I). The differentiating cells develop a neuronal phe notype with neurites and growth cones and sustained activation of prot ein kinase C (PKC) and pp60(c-src). The TPA-induced p130(cas) phosphor ylation increased within 5 min of stimulation and persisted for at lea st 4 days, whereas bFGF/IGF-I-induced p130(cas) phosphorylation was bi phasic, However, the increase in tyrosine phosphorylation of p130(cas) was not restricted to differentiation inducing stimuli. The phosphory lation was blocked by the specific PKC inhibitor GF 109203X, and trans ient transfection with active PKC-epsilon induced p130(cas) tyrosine p hosphorylation, pp60(c-src), known to directly phosphorylate p130(cas) in other cell systems, was not activated after stimulation with TPA o r bFGF/IGF-I for up to 30 min, and the initial p130(cas) phosphorylati on was resistant to the Src family kinase inhibitor herbimycin A. Howe ver, in long term stimulated cells, herbimycin A blocked the induced p hosphorylation of p130(cas), Also, overexpression of src induced phosp horylation of p130(cas). p130(cas) protein and phosphorylated p130(cas ) were present in growth cones isolated from differentiated SH-SY5Y ce lls. Inhibition of PKC activity in differentiating cells with GF 10920 3X 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.