Substrates for protein kinase C in cytosol of EL4 mouse thymoma cells.
Phorbol esters trigger production of interleukin 2 by EL4 thymoma cells via an interaction with specific receptors, now considered to be identical with protein kinase C. Several in vitro substrates for protein kinase C were characterized by incubating cytosol from phorbol ester-responsive and -nonresponsive cells with [32P]adenosine triphosphate and CaCl2 with or without phosphatidylserine and diolein and separating proteins by gel electrophoresis. Phosphorylation of these proteins was calcium dependent in the range of 1-100 microM and stimulated by 10-150 micrograms of phosphatidylserine per ml. Calcium concentrations above 500 microM inhibited 32P incorporation and decreased phospholipid stimulation. Phorbol-12-myristate-13-acetate stimulated phosphorylation of these proteins, with a maximal concentration of 10 nM, providing strong evidence that these are protein kinase C substrates. The substrates for protein kinase C coeluted with the enzyme after binding to a phosphatidylserine affinity column in a calcium-dependent manner. Molecular weights of the protein kinase C substrates in sensitive cell cytosol were approximately 92,000, 84,000, 70,000, 67,000, 53,000, 45,000, 40,000, 36,000, and 20,000. A similar EL4 line which has phorbol ester receptors and protein kinase C, but does not produce interleukin 2 in response to phorbol esters, lacked the Mr 45,000 substrate and often also lacked the Mr 40,000 and 36,000 substrates. These proteins were also analyzed by two-dimensional electrophoresis. These results provide evidence of differences in the two cell lines in the ability of some proteins to serve as substrates for protein kinase C. Four proteins in a highly purified preparation of protein kinase C, at molecular weights of 66,000, 74,000, and 78,000 (all with pI 6.5-7.1) and of 62,000 (pI 6.2-6.4), were protein kinase C substrates, one of which is probably protein kinase C.
C M
CM
Kramer
J J
JJ
Sando
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000032:analysis*; D051379:Mice; D008970:Molecular Weight; D010703:Phorbol Esters / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013945:Thymoma / Q000032:analysis*; D013953:Thymus Neoplasms / Q000032:analysis*
D010703:Phorbol Esters; D010743:Phospholipids; D010750:Phosphoproteins; D011506:Proteins; D011493:Protein Kinase C; D002118:Calcium