Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development.

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Côté, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.

Structural analysis of myosin heavy chain kinase A from Dictyostelium. Evidence for a highly divergent protein kinase domain, an amino-terminal coiled-coil domain, and a domain homologous to the beta-subunit of heterotrimeric G proteins.

We report here the cloning and characterization of the gene encoding the 130-kDa myosin heavy chain kinase (MHCK A) from the amoeba Dictyostelium. Previous studies have shown that purified MHCK A phosphorylates threonines in the carboxyl-terminal tail portion of the Dictyostelium myosin II heavy chain and that phosphorylation of these sites is critical in regulating the assembly and disassembly of myosin II filaments in vitro and in vivo. Biochemical analysis of MHCK A, together with analysis of the primary sequence, suggests that the amino-terminal approximately 500 amino acids form an alpha-helical coiled-coil domain and that residues from position approximately 860 to the carboxyl terminus (residue 1146) form a domain with significant similarity to the beta-subunit of heterotrimeric G proteins. No part of the MHCK A sequence displays significant similarity to the catalytic domain of conventional eukaryotic protein kinases. However, both native and recombinant MHCK A displayed autophosphorylation activity following renaturation from SDS gels, and MHCK A expressed in Escherichia coli phosphorylated purified Dictyostelium myosin, confirming that MHCK A is a bona fide protein kinase. Cross-linking studies demonstrated that native MHCK A is a multimer, consistent with the presence of an amino-terminal coiled-coil domain. Southern blot analysis indicates that MHCK A is encoded by a single gene that has no detectable introns.