Zipper‐Interacting Protein Kinase: Inferring Function In Smooth Muscle Contractility By Identifying Bona Fide Substrates

Zipper‐interacting protein kinase (ZIPK) has been implicated in Ca 2+ ‐independent smooth muscle contraction, although its specific role is unknown. To understand the role of ZIPK in smooth muscle contractility, we set out to identify bona fide ZIPK substrates, using a technique that takes advantage of ‘bulky’ ATP analogues (modified at the N 6 position) and a bioengineered ZIPK capable of utilizing such substrates. Conserved amino acid residues in ZIPK that come into close contact with the N 6 region of ATP were identified and mutated. To identify substrates, 32 P‐labeled 6‐Phe‐ATP and ZIPK L93G protein were added to permeabilized rat caudal arterial smooth muscle strips. 32 P‐labelled substrate proteins were detected by autoradiography and phosphorimaging following SDS‐PAGE. Mass spectrometry was employed to identify ZIPK substrates and LC 20 was confirmed as a direct target. Two other substrates (18 kDa and 40 kDa) were also detected. Experiments were also carried out with rat caudal arterial smooth muscle in the absence of endogenous ATP, but supplemented with 6‐Phe‐ATP and ZIPK L93G, and putative ZIPK substrates identified by western blotting with phosphospecific antibodies. LC 20 was confirmed as a direct target of ZIPK by this method; however no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in regulation of smooth muscle contraction through direct phosphorylation of LC 20 .