DIFFERENTIAL RESPONSES OF PROTEIN-KINASE-C SUBSTRATES (MARCKS, NEUROMODULIN, AND NEUROGRANIN) PHOSPHORYLATION TO CALMODULIN AND S100

Phosphorylation of three physiological substrates of protein kinase C (PKC), MARCKS, neuromodulin (Nm), and neurogranin (Ng), was analyzed t o determine their relative efficacy as substrates of PKC alpha, beta, and gamma and sensitivities to inhibition by calmodulin (CaM) and S100 . Comparison of the V-max/K-m of the phosphorylation of each individua l substrate indicated the order of efficacy as PKC substrate was MARCK S > Nm > Ng. Phosphorylation of these proteins in a mixture by PKC bet a and gamma was indistinguishable from that when each individual subst rate was phosphorylated by these two isozymes, In contrast, the rates of PKC alpha-catalyzed phosphorylation of Nm and Ng in a mixture also containing MARCKS were significantly reduced as compared to that when Nm or Ng was individually phosphorylated by this isozyme. When these s ubstrates were present in a mixture, both CaM and S100 inhibited the P KC-catalyzed phosphorylation of MARCKS to a higher degree than that of Nm or Ng. Protease-activated catalytic fragment of PKC (PKM) was used to determine the effects of Ca2+ and phospholipid on the CaM and S100 -mediated inhibition of PKC substrate phosphorylation. CaM and S100 in hibited the PKM-catalyzed phosphorylation of MARCKS only in the presen ce of Ca2+ and addition of phosphatidylserine (PS)/dioleoylglycerol (D G) did not influence the inhibitory effect. Phosphorylation of Nm or N g by PKM was inhibited by CaM to a higher degree in the absence than i n the presence of Ca2+. S100 was ineffective in inhibiting the phospho rylation of Nm and Ng without Ca2+ and only poorly effective in the pr esence of Ca2+. The CaM-mediated inhibition of Nm or Ng phosphorylatio n by PKM was also not affected by PS/DG either with or without Ca2+. T he results presented here demonstrate that MARCKS is a preferred subst rate of PKC and its phosphorylation by PKC is most sensitive to inhibi tion by regulatory proteins such as CaM and S100. (C) 1995 Academic Pr ess, Inc.