Purification and characterization of a Dictyostelium protein kinase required for actin activation of the Mg2+ ATPase activity of Dictyostelium myosin ID.
We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 mumol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD.
Lee
S F
SF
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Côté
G P
GP
D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives; D002852:Chromatography, Ion Exchange; D004023:Dictyostelium / Q000201:enzymology*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism*; D008970:Molecular Weight; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000502:physiology; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000302:isolation & purification* / Q000502:physiology
D000199:Actins; D005656:Fungal Proteins; D007527:Isoenzymes; D015800:Protozoan Proteins; D002482:Cellulose; C008881:phosphocellulose; D011494:Protein Kinases; C093485:110 kDa protein kinase, Dictyostelium; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins