| The regulation of myosins is required for most motile processes. Regulation of the myosin II isoform by its assembly into filaments is achieved by reversible phosphorylation of its C-terminus. Myosin II heavy-chain-specific protein kinase C (MHC-PKC) was shown to be a diacylglycerol kinase. Previous studies disrupting the corresponding gene locus resulted in cells with altered myosin II assembly levels consistent with the loss of a MHCK. This phenotype is instead a consequence of the disruption of dgkA function leading to the possibility that diacylglycerol and/or phosphatidic acid metabolism plays a role in controlling myosin II assembly.; Binding of Ca2+ to light chains can also regulate myosin activity. A novel calmodulin-related light chain, MlcD, was shown to associate with the class I myosin, MyoD, but not to the other two long-tailed Dictyostelium myosin I isozymes, MyoB and MyoC. In vitro analysis indicated its single metal binding site had too low an affinity for M1cD to function as a Ca2+-sensitive light chain. MyoD activity is instead regulated via motor domain phosphorylation by the p21-activated kinase PakB. This kinase was shown to adopt a diffuse intracellular localization but moderately redistributed to sites known to contain myosin I such as the leading edge of migrating cells, pinosomes, phagosomes and small mobile punctae at the rear of chemotaxing cells. Deletion of the PakB Rac-binding domain from the full-length protein resulted in a mutant with constitutive kinase activity which exhibited an increased association with these same structures. Furthermore, the expression of this mutant in Dictyostelium induced defects in cytokinesis and in addition, cells displayed increased rates of phagocytosis and pinocytosis. PakB localization required an intact N-terminus, which was shown to interact with the SH3 domain of the Dictyostelium ortholog of yeast Abp1 (Dabp1). Co-localization and co-immunoprecipitation experiments supported an association between DABP1 and the active, but not inactive, form of PAR Dicryostelium in which the abp gene was disrupted had a cytokinesis defect and in addition displayed increased rates of phagocytosis but not pinocytosis. These results support a model whereby PakB activation by Racs is coupled to its association with Dabp1 at sites of dynamic actin assembly. |
