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

Biochemistry. 2000 Sep 19;39(37):11441-51. doi: 10.1021/bi000347e.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / chemistry
  • Actin Cytoskeleton / genetics
  • Actin Cytoskeleton / metabolism
  • Amino Acid Sequence
  • Animals
  • Aspartic Acid / genetics
  • Brain / metabolism
  • Casein Kinase II
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Myosin Heavy Chains / antagonists & inhibitors
  • Myosin Heavy Chains / chemistry
  • Myosin Heavy Chains / genetics
  • Myosin Heavy Chains / metabolism*
  • Peptide Fragments / antagonists & inhibitors
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Protein Binding / genetics
  • Protein Isoforms / antagonists & inhibitors
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Kinase C / metabolism
  • Protein Serine-Threonine Kinases / metabolism
  • Rabbits
  • S100 Proteins / metabolism*
  • S100 Proteins / physiology
  • Sodium Chloride / pharmacology


  • Peptide Fragments
  • Protein Isoforms
  • S100 Proteins
  • Aspartic Acid
  • Sodium Chloride
  • Casein Kinase II
  • Protein Serine-Threonine Kinases
  • Protein Kinase C
  • Myosin Heavy Chains