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Zipper-interacting protein kinase (ZIPK) has been implicated in Ca2؉ -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 .Smooth muscle plays an important role in the regulation of diverse physiological processes, including vascular tone, gastrointestinal motility, penile erection, bronchial diameter, and parturitional/postparturitional myometrial contraction. All smooth muscle tissues rely on the Ca 2ϩ /calmodulin-dependent activation of myosin light chain kinase (MLCK) 6 and subsequent phosphorylation of the 20-kDa myosin regulatory light chains (LC 20 ) at Ser-19 to initiate actomyosin cross-bridge cycling and force development (1). On the other hand, relaxation is induced by dephosphorylation of LC 20 by myosin light chain phosphatase (MLCP), a type 1 protein serine/threonine phosphatase (2, 3). Contraction of multiple smooth muscle tissues has frequently been observed in the absence of an increase in cytosolic free Ca 2ϩ concentration in response to a variety of stimuli (4). This phenomenon, commonly referred to as Ca 2ϩ sensitization, involves alteration of the MLCK:MLCP activity ratio in favor of the kinase, which can be achieved by the following mechanisms: (i) activation of MLCK by a mechanism not involving Ca 2ϩ /calmodulin, e.g. phosphorylation of MLCK by proline-directed kinases (5, 6); (ii) an increase in Ca 2ϩ -independent LC 20 kinase activity (7); and (iii) inhibition of MLCP either directly by phosphorylation of inhibitory residues (Thr-697 and/or Thr-855) in the myosin phosphatase targeting subunit 1 (MYPT1) of MLCP (8 -10) or indirectly by phosphorylation of the protein kinase C-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI-17) at Thr-38 (11, 12). The observation that intact and permeabilized smooth muscle tissues exhibit Ca 2ϩ -independent contracti...

2ϩsensitization in rat ileal longitudinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 293: G699-G710, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00214.2007.-We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca 2ϩ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 M microcystin induced LC20 diphosphorylation and contraction of ␤-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 M, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca 2ϩ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle. ileum; protein kinase C; myosin phosphatase; mitogen-activated protein kinase; protein kinase C-potentiated phosphatase inhibitor protein of 17 kDa; myosin-targeting subunit of myosin light chain phosphatase SMOOTH MUSCLE CONTRACTION is a dynamic and highly regulated process. The contractile state of smooth muscle is mainly regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC 20 ] i )} is the primary determinant of smooth muscle contraction. Force can be further increased through signaling pathways that modulate MLCK and/or MLCP activities. The Ca 2ϩ sensitivity of contraction can be affected by any change in the ratio of MLCK:MLCP activity. A decrease in MLCP activity will shift the balance in favor of MLCK, resulting in a greater degree of LC 20 phosphorylation and contraction. The phenomenon of ...

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Ca 2ϩ -independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide. Am J Physiol Gastrointest Liver Physiol 297: G361-G370, 2009. First published June 18, 2009 doi:10.1152/ajpgi.00112.2009.-As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Tritonskinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca 2ϩ -independent, microcystin-and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca 2ϩ -independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC 20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca 2ϩ -independent LC 20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1-320) -D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC 20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility. Although ZIPK has been linked to the regulation of smooth muscle contraction, it is unclear whether its role in these processes is attributable solely to the direct phosphorylation of LC 20 or the inhibition of MLCP via phosphorylation of MYPT1. A specific inhibitor of ZIPK would be an important tool for the delineation of its physiological function in smooth muscle contraction. Unfortunately, small molecule inhibitors that are selective for ZIPK have yet to be developed. Small molecule inhibitors of ZIPK (e.g., ML-9, ML-7, staurosporine, and wortmannin) (2) are also active against a number of other protein kinases found in smooth muscle. The nonselec...