Mutagenesis analysis of human SM22: Characterization of actin binding

Y. Fu, H. W. Liu, S. M. Forsythe, P. Kogut, J. F. McConvllle, A. J. Halayko, B. Camoretti-Mercado, J. Solway

Research output: Contribution to journalArticlepeer-review

107 Scopus citations


SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH2-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH2-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.

Original languageEnglish (US)
Pages (from-to)1985-1990
Number of pages6
JournalJournal of applied physiology
Issue number5
StatePublished - 2000


  • Arterial
  • Asthma
  • Gene
  • Smooth muscle
  • Vascular

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)


Dive into the research topics of 'Mutagenesis analysis of human SM22: Characterization of actin binding'. Together they form a unique fingerprint.

Cite this