Specific phosphorylation by calcium-EGTA complex of a 75 kDa human tumor plasma membrane protein (pp75).
1. The major functional role played by phosphorylation of plasma membrane proteins in the biological properties of tumor cells suggests that identification of protein kinases and their substrates will contribute to our understanding of the molecular basis of the malignant process and of the aberrant behavior of tumor cells. 2. The present study has investigated the phosphorylation of surface proteins of human tumor cells. Incubation of plasma membranes isolated from cultured human melanoma cells with [gamma-32P]ATP in the presence of Ca2+ and ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) resulted in specific phosphorylation of serine and threonine residues on a 75kDa protein (pp75). 3. Neither Ca2+ or EGTA alone, nor any other divalent metal ion tested could induce phosphorylation of pp75. 4. The phosphorylation of pp75 was directly dependent upon the presence of non-ionic detergents, and was influenced by length of incubation and concentration ratio of Ca2+ and EGTA. 5. Incubation of isolated plasma membranes with [gamma-32P]ATP in the presence of Ca2+ and EGTA and immunochemical analysis by Western blotting with an anti pp75 xenoantiserum detected the pp75 in human melanoma, neuroblastoma, ovarian carcinoma and lymphoid T cells and fibroblasts but not in B-lymphoid cells, renal carcinoma cells, peripheral blood lymphocytes and splenocytes. 6. These results suggest the presence of a new class of plasma membrane bound protein kinases activated by chelated calcium and differentially expressed in normal and transformed human cells.
Ziai
M R
MR
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.
Ferrone
S
S
D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D002855:Chromatography, Thin Layer; D004533:Egtazic Acid / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D009363:Neoplasm Proteins; D004533:Egtazic Acid; D002118:Calcium