Mutagenesis analysis of human SM22: characterization of actin binding

J Appl Physiol (1985). 2000 Nov;89(5):1985-90. doi: 10.1152/jappl.2000.89.5.1985.


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 NH(2)-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 NH(2)-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / analysis
  • Actins / metabolism*
  • Animals
  • Asthma / metabolism
  • Calcium / pharmacology
  • Humans
  • Microfilament Proteins*
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics*
  • Muscle Proteins / metabolism*
  • Muscle, Smooth / chemistry*
  • Muscle, Smooth / enzymology
  • Mutagenesis / physiology
  • Phosphorylation
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Protein Kinase C / metabolism
  • Protein Structure, Tertiary
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Serine / metabolism
  • Trachea / chemistry
  • Trachea / enzymology


  • Actins
  • Microfilament Proteins
  • Muscle Proteins
  • Recombinant Proteins
  • Tagln protein, mouse
  • transgelin
  • Serine
  • Protein Kinase C
  • Calcium