Dictyostelium myosin-II heavy-chain kinase A is activated by autophosphorylation: studies with Dictyostelium myosin-II and synthetic peptides

• Published in: Biochemistry (Easton) Vol. 29; no. 38; pp. 8992 - 8997
• Main Authors: Medley, Quintus G, Gariepy, Jean, Cote, Graham P
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 25.09.1990
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Calcium-Calmodulin-Dependent Protein Kinases; Dictyostelium - enzymology; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular Sequence Data; Myosin Subfragments - pharmacology; Phosphorylation - drug effects; Phosphotransferases - metabolism; Protozoan Proteins; Substrate Specificity; Transferases; Dictyostelium discoideum; Protozoa; Enzymatic activity; Peptides; Enzyme; Protein kinase; Myosin; Contractile protein; Activation; Kinetic parameter; Chemical synthesis; Mycetozoa
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00490a016

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.