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.
Futey
L M
LM
Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, Ohio 44106.
Medley
Q G
QG
Côté
G P
GP
Egelhoff
T T
TT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004023:Dictyostelium / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015800:Protozoan Proteins; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D019204:GTP-Binding Proteins