An analysis of the mechanism of Dictyostelium myosin heavy chain kinase B in substrate targeting

Previous studies have shown that myosin heavy chain kinase A and B (MHKA and MHKB) are able to phosphorylate and disassemble Dictyostelium discoideum myosin II bipolar filaments by directly targeting myosin II via a conserved region of WD repeats, also referred to as the WD repeat domain. MHKA lacking this domain is unable to phosphorylate myosin II. By comparison, MHKB lacking this same domain is able to phosphorylate myosin II, albeit at a significantly reduced level compared to the full-length protein. The main difference between the MHKA and MHKB proteins is the presence of a region of MHKB in which the amino acid asparagine is repeated 25 times, called the asparagine-rich (pN) region. The function of this region has not been well studied. The research described in this thesis focuses on the asparagine-rich and WD repeat domains of MHKB and their roles in targeting myosin II for phosphorylation. My studies revealed that overexpressing full-length MHKB in Dictyostelium cells resulted in slowed growth rates due to cytokinesis defects in which cells become large and multinucleated. MHKB lacking the WD repeat domain (MHKB-DeltaWD) also showed this large multinucleated phenotype and slowed growth rate. However, cells overexpressing a truncation of MHKB lacking both the WD and pN regions (MHKB-DeltapN-WD) grew normally and did not become multi-nucleated; indicating that the WD repeat domain is not the only targeting region of MHKB. Triton X-100 cytoskeleton fractionation assays revealed that cells overexpressing full-length MHKB or MHKB-DeltaWD have more myosin II in the cytosol than in the cytoskeleton, which is indicative of myosin II being phosphorylated and disassembled by these two versions of MHKB. By contrast, the myosin II in cells overexpressing the MHKB-DeltapN-WD truncation was located primarily in the cytoskeletal fraction, suggesting that the poly-asparagine region of MHKB may be involved in targeting the kinase to phosphorylate myosin II heavy chain (MHC). Biochemical assays of kinase activity toward myosin II substrate revealed that while full-length MHKB and the MHKB-DeltaWD truncation were able to phosphorylate myosin II, the activity of the MHKB-DeltapN-WD truncation toward myosin II substrate was below the limits of detection. This is despite the fact that all three versions of the kinase phosphorylate a peptide substrate to essentially the same level. In summary, MHKB has two major regions involved in targeting myosin II for phosphorylation. The WD-repeat domain is known to directly target myosin II, but is not necessary for the kinase to function. My studies of this system have revealed that the pN region, in combination with the WD-repeat domain, is necessary for MHKB to target myosin II for phosphorylation.