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 (MIIA(F46) and MIIBalpha F47) 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 MIIBalpha F47 [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 (MIIBalpha F47-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg2+, by significantly increasing the critical concentrations for assembly. Without Mg2+, MIIBalpha F47-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIBalpha F47 by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg2+, MIIBalpha F47-wt promoted assembly of MIIBalpha F47-PK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIBalpha F47-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIBalpha F47-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca2+-dependent manner to MIIA(F46), but not appreciably to MIIBalpha F47. At 0.15 M NaCl, mts 1-Ca2+ not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIBalpha F47 in, MIIA(F46) and MIIBalpha F47 mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MW and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.