Protein kinase C-dependent tyrosine phosphorylation of p130cas in differentiating neuroblastoma cells.
The cell signaling docking protein p130cas 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 pp60c-src. The TPA-induced p130cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130cas 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 p130cas tyrosine phosphorylation. pp60c-src, known to directly phosphorylate p130cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130cas. Also, overexpression of src induced phosphorylation of p130cas. p130cas protein and phosphorylated p130cas 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 p130cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130cas. These data suggest a function for p130cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.
Fagerström
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Department of Laboratory Medicine, Lund University, University Hospital MAS, Malmö, Sweden.
Påhlman
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Nånberg
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D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D050719:Crk-Associated Substrate Protein; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009447:Neuroblastoma / Q000473:pathology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D003994:Bucladesine; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; C070515:bisindolylmaleimide I