Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP.
The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003208:Concanavalin A / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013154:Spleen / Q000166:cytology
D008244:Lysophosphatidylcholines; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D003208:Concanavalin A; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium