The structure and phosphorylation of two protein kinase C (PKC) α substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The α-peptide, which exhibits the typical PKC α substrate motif and is based on the pseudosubstrate region of PKC α, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the α-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.
Influence of lipid on the structure and phosphorylation of protein kinase C α substrate peptides
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B. Betsy VINTON, L. Stacey WERTZ, Jaison JACOB, Joanna STEERE, M. Charles GRISHAM, S. David CAFISO, J. Julianne SANDO; Influence of lipid on the structure and phosphorylation of protein kinase C α substrate peptides. Biochem J 15 March 1998; 330 (3): 1433–1442. doi: https://doi.org/10.1042/bj3301433
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