title: Dictyostelium myosin II heavy-chain kinase A is activated by autophosphorylation: studies with Dictyostelium myosin II and synthetic peptides.
abstract: One of the major sites phosphorylated on the Dictyostelium myosin II heavy chain by the Dictyostelium myosin II heavy-chain kinase A (MHCK A) is Thr-2029. Two synthetic peptides based on the sequence of the Dictyostelium myosin II heavy chain around Thr-2029 have been synthesized: MH-1 (residues 2020-2035; RKKFGESEKTKTKEFL-amide) and MH-2 (residues 2024-2035). Both peptides are substrates for MHCK A and are phosphorylated to a level of 1 mol of phosphate/mol. Tryptic digests indicate that the peptides are phosphorylated on the threonine corresponding to Thr-2029. When assays are initiated by the addition of MHCK A, the rate of phosphate incorporation into the peptides increases progressively for 4-6 min. The increasing activity of MHCK A over this time period is a result of autophosphorylation. Although each 130-kDa subunit of MHCK A can incorporate up to 10 phosphate molecules, 3 molecules of phosphate per subunit are sufficient to completely activate the kinase. Autophosphorylated MHCK A displays Vmax values of 2.2 and 0.6 mumol.min-1.mg-1 and Km values of 100 and 1200 microM with peptides MH-1 and MH-2, respectively. Unphosphorylated MHCK A displays a 50-fold lower Vmax with MH-1 but only a 2-fold greater Km. In the presence of Dictyostelium myosin II, the rate of autophosphorylation of MHCK A is increased 4-fold. If assays are performed at 4 degrees C (to slow the rate of MHCK A autophosphorylation), autophosphorylation can be shown to increase the activity of MHCK A with myosin II.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: Q G
authors.initials: QG
authors.lastname: Medley
authors.forename: J
authors.initials: J
authors.lastname: Gariépy
authors.forename: G P
authors.initials: GP
authors.lastname: Côté
mesh_terms: D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004023:Dictyostelium / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010770:Phosphotransferases / Q000378:metabolism*; D015800:Protozoan Proteins; D013379:Substrate Specificity
chemical_list: D015879:Myosin Subfragments; D015800:Protozoan Proteins; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase