Structure-function relations of smooth muscle calponin. The critical role of serine 175.
Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin-dependent protein kinase II. To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II. The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively). Tissue-purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective. Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases. These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate.
Tang
D C
DC
Smooth Muscle Research Group and the Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
Kang
H M
HM
Jin
J P
JP
Fraser
E D
ED
Walsh
M P
MP
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D018604:Epitope Mapping; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017931:DNA Primers; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009218:Myosins; D011493:Protein Kinase C; D011994:Recombinant Proteins; D012694:Serine; D000096985:Calponins