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1 Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada
2 School of Molecular & Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia
3 Department of Medical Biochemistry, University of Calgary, Calgary, Canada; Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada
* To whom correspondence should be addressed. E-mail: jmacdo{at}ucalgary.ca.
As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) phosphorylates the regulatory myosin light chain (RLC20) at Ser19 and Thr18 in a Ca2+-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to sequence found in ZIPK. The sequence similarity prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of the constitutively active, 32 kDa form of ZIPK with an apparent Ki value of 3.4 µM. The SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of non-specific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK (and ILK) and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues.
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