Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA.

In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIAF46 and MIIBαF47) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIBαF47 [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIBαF47-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg2+, by significantly increasing the critical concentrations for assembly. Without Mg2+, MIIBαF47-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIBαF47 by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg2+, MIIBαF47-wt promoted assembly of MIIBαF47-CK-5D and -PK-4D in homofragment mixtures, but not by forming hete...