Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides.
Phosphorylation of a single threonine (myosin IA) or serine (myosins IB and IC) in the heavy chains of the Acanthamoeba myosin I isozymes is required for expression of their actin-activated Mg2(+)-ATPase activities. We now report that the synthetic peptide Gly-Arg-Gly-Arg-Ser-Ser-Val-Tyr-Ser, which corresponds to the phosphorylated region of Acanthamoeba myosin IC, is a good substrate for myosin I heavy chain kinase: Km = 54 microM, and Vmax = 15 mumols/min.mg. The same serine is phosphorylated as in the native substrate (residue 6 in the above sequence), and kinase activity with the synthetic peptide as substrate is also stimulated by phosphatidylserine-enhanced autophosphorylation of the kinase. These results indicate that all of the essential sequence determinants of kinase specificity are contained within this 9-residue peptide. With the peptide as substrate, we found that another acidic phospholipid, phosphatidylinositol, also enhances autophosphorylation of the kinase whereas the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine do not. By comparing the Km and Vmax values for a series of synthetic peptide substrates, we established that 1 basic amino acid is essential on the NH2-terminal side of the phosphorylation site, and two are preferable, and that a tyrosine is essential 2 residues away on the COOH-terminal side. There is a slight preference for arginines over lysines. All of these local sequence specificity determinants are present in the three native substrates, Acanthamoeba myosins IA, IB, and IC, and in two Dictyostelium myosin I isozymes that are putative substrates for the kinase. Similar sequences do not occur in the myosins I from intestinal brush border, which is not a substrate for the Acanthamoeba kinase.
Brzeska
H
H
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Lynch
T J
TJ
Martin
B
B
Corigliano-Murphy
A
A
Korn
E D
ED
D000048:Acanthamoeba / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D015800:Protozoan Proteins; D013379:Substrate Specificity
D009842:Oligopeptides; D010718:Phosphatidylserines; D015800:Protozoan Proteins; D004533:Egtazic Acid; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D002118:Calcium