Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase.
The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.
Brzeska
H
H
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Martin
B
B
Kulesza-Lipka
D
D
Baines
I
I
Korn
E D
ED
D000048:Acanthamoeba / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002384:Catalysis; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins; D014357:Trypsin / Q000378:metabolism
D010743:Phospholipids; D015800:Protozoan Proteins; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D002918:Chymotrypsin; D014357:Trypsin