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.