Mutagenesis analysis of human SM22: characterization of actin binding.
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.
Fu
Y
Y
Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Liu
H W
HW
Forsythe
S M
SM
Kogut
P
P
McConville
J F
JF
Halayko
A J
AJ
Camoretti-Mercado
B
B
Solway
J
J
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001249:Asthma / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D006801:Humans; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009130:Muscle, Smooth / Q000737:chemistry* / Q000201:enzymology; D016296:Mutagenesis / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014132:Trachea / Q000737:chemistry / Q000201:enzymology
D000199:Actins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; C058992:Tagln protein, mouse; C051615:transgelin; D012694:Serine; D011493:Protein Kinase C; D002118:Calcium