STIMULATION BY GLIA MATURATION FACTOR OF CA-2+-DEPENDENT PHOSPHORYLATION OF MR 100-K PROTEIN IN RAT GLIOBLASTS

When quiescent rat glioblasts were stimulated by glia maturation factor (GMF), their intrinsic Ca2+-dependent phosphorylation of proteins, especially that of Mr 100 k protein, increased. The phosphorylation of Mr 100 k protein in the homogenate started rising 13 h (S phase) after GMF stimulation and reached the maximal level (8-fold greater than the control) at 26 h. Phosphorylation was also detected in intact cells by the use of [32P]orthophosphate. Calmodulin augmented and W-7 (calmodulin inhibitor) slightly inhibited the phosphorylation, suggesting that Ca2+/calmodulin-dependent protein kinase may partly be involved in phosphorylation of the Mr 100 k protein. Subcellular fractionation experiments revealed that both Mr 100 k protein and its kinase were localized exclusively in the cytosol. We also found marked phosphorylation of Mr 100 k protein in neural tumor cell lines, mouse neuroblastoma (Neuro2a and NAs-1) and glioma (C6 and 354A). Since the peptide maps of 32P-labeled peptides obtained by chemical cleavage from Mr 100 k protein of the cells were identical to those of glioblasts, the Mr 100 k proteins, regardless of cell origin, may be closely related in structure. Growth inhibitors, W-7 (50 μM), puromucin (2 μM), spongoadenosine (50 μM), diphenylhydantoin (0.3 mM), α-sialosyl cholesterol (20 μg/ml) and protein kinase inhibitor, K252a (50 nM), lowered the phosphorylation of the Mr 100 k protein in the cell homogenate derived from glioblasts pretreated with the drugs for 24 h. Mr 100 k protein of glioblasts and C6 cells was immunoprecipitated by anti-elongation factor-2 (EF-2) antiserum indicating an identity or similarity in structure between the protein and EF-2. These findings provide a possibility that cell growth may be brought about through a phosphorylation of Mr 100 k protein as one of the signal transduction processes subsequent to a mitogen stimulation. © 1990.