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 MIIB(alpha)(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 MIIB(alpha)(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 (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(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 MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.
Murakami
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N
Laboratories of Neurobiochemistry, Molecular Neurobiology, and Molecular Regulations, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA. pjisadog@interport.net
Kotula
L
L
Hwang
Y W
YW
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001921:Brain / Q000378:metabolism; D047390:Casein Kinase II; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011817:Rabbits; D009418:S100 Proteins / Q000378:metabolism* / Q000502:physiology; D012965:Sodium Chloride / Q000494:pharmacology
D010446:Peptide Fragments; D020033:Protein Isoforms; D009418:S100 Proteins; D001224:Aspartic Acid; D012965:Sodium Chloride; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D018995:Myosin Heavy Chains