title: Regulation of peptide-calmodulin complexes by protein kinase C in vivo.
abstract: We used the freshwater protozoan Paramecium tetraurelia to investigate the potential regulation by protein kinase C of calmodulin interactions with binding peptides in intact cells. In these organisms, an action potential results in membrane depolarization and a period of backward swimming; repolarization and a return to forward swimming requires the presence of normal calmodulin. We postulated that injection of high-affinity calmodulin binding peptides might interfere with repolarization and thus prolong the period of membrane depolarization. Synthetic peptides spanning the protein kinase C phosphorylation site/calmodulin-binding domains of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (also known as F52 or MacMARCKS) were injected into cells; these caused a 2- to 3-fold increase in the duration of backward swimming. Similar changes were seen with two other calmodulin-binding peptides. This behavioral response could be prevented by coinjecting calmodulin. Activation of Paramecium protein kinase C with an active phorbol ester completely reversed (within 3 min) the behavioral effects of the normal MARCKS and MARCKS-related protein peptides. Injection of a nonphosphorylatable peptide, in which alanines were substituted for serines, resulted in the usual behavioral response; however, this was not reversed by phorbol ester treatment. The corresponding aspartate-substituted peptide, which has a 10-fold lower affinity for calmodulin, did not prolong backward swimming. These data suggest that these peptides can form complexes with calmodulin at the calcium concentrations that prevail in intact Paramecium cells and that such complexes can be disrupted by protein kinase C phosphorylation of the peptides.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
authors.forename: R D
authors.initials: RD
authors.lastname: Hinrichsen
authors.forename: P J
authors.initials: PJ
authors.lastname: Blackshear
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009068:Movement / Q000187:drug effects; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009842:Oligopeptides / Q000494:pharmacology; D016805:Paramecium tetraurelia / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate