Dictyostelium myosin heavy chain kinase A subdomains. Coiled-coil and wd repeat roles in oligomerization and substrate targeting.
Myosin heavy chain kinase A (MHCK A) participates in the regulation of cytoskeletal myosin assembly in Dictyostelium, driving filament disassembly via phosphorylation of sites in the myosin tail. MHCK A contains an amino-terminal coiled-coil domain, a novel central catalytic domain, and a carboxyl-terminal domain containing a 7-fold WD repeat motif. We have overexpressed MHCK A truncation constructs to clarify the roles of each of these domains. Recombinant full-length MHCK A, MHCK A lacking the predicted coiled-coil domain, and MHCK A lacking the WD repeat domain were expressed at high levels in Dictyostelium cells lacking endogenous MHCK A. Biochemical analysis of the purified proteins demonstrates that the putative coiled-coil domain is responsible for the oligomerization of the MHCK A holoenzyme. Removal of the WD repeat domain had no effect on catalytic activity toward a synthetic peptide, but did result in a 95% loss of protein kinase activity when native myosin filaments were used as the substrate. Cellular analysis confirms that the same severe loss of activity against myosin occurs in vivo when the WD repeat domain is eliminated. These results suggest that the WD repeat domain of MHCK A serves to target this enzyme to its physiological substrate.
Kolman
M F
MF
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Egelhoff
T T
TT
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002455:Cell Division; D004023:Dictyostelium; D007700:Kinetics; D008970:Molecular Weight; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins; D016626:Shadowing Technique, Histology; D013379:Substrate Specificity
D011108:Polymers; D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D009218:Myosins