Abstract
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.
Highlights
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
Recombinant fulllength MHC kinases (MHCKs) A is expressed at 27 mM, DCoil-MHCK A is expressed at 46 mM, and DWD-MHCK A is expressed at 90 mM
Gel filtration analysis of the purified constructs demonstrated a loss of oligomerization in the DCoil-MHCK A construct
Summary
(Received for publication, February 11, 1997, and in revised form, April 3, 1997). From the Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970. The 3X ASP cells are phenotypically identical to MHC null cells (mhc2) These studies indicate that the MHCK A target sites play a critical role in regulated myosin II assembly in vitro and that filament assembly is required for myosin function in vivo. Subsequent cellular analysis of Dictyostelium MHCK A null cells (mhck A2) and overexpressing cell lines (MHCK A1) indicated that MHCK A regulates the cytoskeletal myosin II assembly level during both growth and development [14]. Molecular analysis of the MHCK A sequence [15] indicates that it has an amino-terminal domain that is predicted to have an a-helical coiled-coil structure, a catalytic domain that is not related to conventional protein kinases, and a carboxyl-terminal domain that contains the 7-fold WD repeat motif [16, 17] characteristic of b-subunits of heterotrimeric G proteins.
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