Structure-Function Relations of Smooth Muscle Calponin
THE CRITICAL ROLE OF SERINE 175 (*)
- From the 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
- §§Medical Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Medical Biochemistry, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3021; Fax: 403-270-2211; walsh{at}acs.ucalgary.ca.
Abstract
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 (K
= 0.8, 1.3, and 1.0 μM, respectively), whereas the affinities for S175A and S175D calponins were much lower (K
= 26.8 and 44.2 μM, 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.
Footnotes
-
↵¶ Recipient of a Heart and Stroke Foundation of Canada Fellowship.
-
↵** Recipient of a Heart and Stroke Foundation of Canada Scholarship.
-
↵* This work was supported by grants (to J.-P. J. and M. P. W.) from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- PKC
-
Ca
- and phospholipid-dependent protein kinase C
- CaM kinase II
-
Ca
/calmodulin-dependent protein kinase II
- MOPS
-
3-(N-morpholino)propanesulfonic acid
- PBS-T
-
phosphate-buffered saline containing Tween-20
- PCR
-
polymerase chain reaction
- PAGE
-
polyacrylamide gel electrophoresis.
-
↵2Jin, J.-P., Walsh, M. P., Resek, M. E., and McMartin, G. A.(1996) Biochem. Cell Biol.74, in press.
-
- Received September 7, 1995.
- Revision received February 2, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
