Chemical Genetics of Zipper-interacting Protein Kinase Reveal Myosin Light Chain as a Bona Fide Substrate in Permeabilized Arterial Smooth Muscle

Smooth Muscle Research Group and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4Z6, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2011; 286(42):36978-91. DOI: 10.1074/jbc.M111.257949
Source: PubMed


Zipper-interacting protein kinase (ZIPK) has been implicated in Ca(2+)-independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC(20) and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC(20) and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N(6) position and an engineered ZIPK capable of utilizing such substrates. (32)P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC(20) as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC(20) was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC(20).

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    • "It has long been recognized that kinases other than MLCK may operate in SM, such as the Ca2+-independent kinases zipper-interacting protein kinase (ZIPK) [32], [35] and the integrin-linked kinase (ILK) [36], [37]. An interesting variation of this scheme involves Ca2+-independent kinases, such as proline-directed kinases [38], [39], [40], acting indirectly on RLC20, by phosphorylating and up-regulating MLCK. "
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