Abstract
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.
Highlights
P130cas is a recently identified docking protein that contains an SH3 domain and a region with several tyrosine residues that can become tyrosine-phosphorylated and constitutes putative SH2 binding domains (1)
It is established that p130cas becomes heavily tyrosine-phosphorylated after integrin stimulation (4 – 6) and that the protein is localized to focal adhesions (4, 7) and along stress fibers (4). p130cas associates with focal adhesion kinase (FAK)1 (7, 8) and pp60c-src (1, 9, 10), both proteins involved in focal adhesion regulation
Immunoprecipitation of p130cas followed by immunoblotting with an anti-phosphotyrosine antibody revealed that in TPA-treated SH-SY5Y cells, p130cas tyrosine phosphorylation was induced within 5 min and remained elevated for at least 4 days (Fig. 1B, upper panel)
Summary
Cell Culture and Reagents—The human neuroblastoma cell line SHSY5Y (25, 26) was cultured in Eagle’s minimum essential medium supplemented with 10% FCS (Life Technologies, Inc.), 100 IU/ml penicillin, and 50 g/ml streptomycin in an atmosphere of 5% CO2 at 37 °C. 2 ϫ 106 SH-SY5Y cells/10-cm dish were plated in FCS-containing Eagle’s medium 24 h before transfection and received fresh medium 3 h prior to transfection. Cell lysates were immunoprecipitated with 1 g of a polyclonal anti-p130cas antiserum (Santa Cruz) and protein A-Sepharose (Pharmacia), and the proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (29) followed by blotting to Hybond C extra filters (Amersham Corp.) (30). Src Kinase Assay—Cells were lysed in RIPA as described above, and pp60c-src was immunoprecipitated with mAb 327 monoclonal anti-Src antibody Subcellular Fractionation—Differentiated SH-SY5Y cells were fractionated into growth cones and cell bodies as described previously (17). The cells were kept in serum-containing medium until additions of TPA or growth factors, when the cultures receiving bFGF/IGF-I were changed to SHTE medium.
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