The catalytic domain of Acanthamoeba myosin I heavy chain kinase. I. Identification and characterization following tryptic cleavage of the native enzyme.
The actin-activated Mg2+-ATPase activities of the myosin I isoenzymes from Acanthamoeba castellanii are greatly increased by phosphorylation catalyzed by myosin I heavy chain kinase (MIHC kinase), a monomeric 97-kDa protein whose activity is greatly enhanced by acidic phospholipids and by autophosphorylation of multiple sites. In this paper, we show that the 35-kDa COOH-terminal fragment obtained by trypsin cleavage of maximally activated, autophosphorylated kinase retains the full activity and two to three of the autophosphorylation sites of the native enzyme. Other autophosphorylation sites occur in the middle third of the native enzyme. A trypsin cleavage site within the 35-kDa region is protected in phosphorylated kinase but is readily cleaved in unphosphorylated kinase producing catalytically inactive 25- and 11-kDa fragments from the NH2- and COOH-terminal ends, respectively, of the 35-kDa peptide. This implies that the conformation around the "25/11" cleavage site changes upon phosphorylation of the native enzyme. The position of this site corresponds to the activation loop of protein kinase A (see the accompanying paper: Brzeska, H., Szczepanowska, J., Hoey, J., and Korn, E. D. (1996) J. Biol. Chem. 271, 27056-27062). Exogenously added MIHC kinase phosphorylates the 11-kDa fragment, but not the 25-kDa fragment, indicating that the phosphorylation sites of the 35-kDa catalytic fragment are located within the COOH-terminal 11 kDa. The accompanying paper describes the cloning, sequencing, and expression of a fully active 35-kDa catalytic domain.
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
H
H
Brzeska
B M
BM
Martin
E D
ED
Korn
D000048:Acanthamoeba / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015800:Protozoan Proteins; D014357:Trypsin / Q000378:metabolism
D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D014357:Trypsin