title: Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.
abstract: 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).
authors: Smooth Muscle Research Group and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4Z6, Canada.
authors: Lori D
authors: LD
authors: Moffat
authors: Shannon B A
authors: SB
authors: Brown
authors: Michael E
authors: ME
authors: Grassie
authors: Annegret
authors: A
authors: Ulke-Lemée
authors: Laura M
authors: LM
authors: Williamson
authors: Michael P
authors: MP
authors: Walsh
authors: Justin A
authors: JA
authors: MacDonald
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001158:Arteries / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D064550:Death-Associated Protein Kinases; D008297:Male; D009119:Muscle Contraction / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D020125:Mutation, Missense; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D051017:Apoptosis Regulatory Proteins; D018994:Myosin Light Chains; D000255:Adenosine Triphosphate; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1