DICTYOSTELIUM MYOSIN-II HEAVY-CHAIN KINASE-A IS ACTIVATED BY AUTOPHOSPHORYLATION - STUDIES WITH DICTYOSTELIUM MYOSIN-II AND SYNTHETIC PEPTIDES

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 μmol-min−1·mg−1 and Km values of 100 and 1200 μM 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 °C (to slow the rate of MHCK A autophosphorylation), autophosphorylation can be shown to increase the activity of MHCK A with myosin II. © 1990, American Chemical Society. All rights reserved.