Ca²⁺-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

Abstract

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...

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

“…We showed previously that Ca 2ϩ -independent, microcystin (phosphatase inhibitor)-induced contraction of Triton-skinned rat caudal arterial smooth muscle strips is not blocked by pretreatment with AV25, suggesting that ZIPK activity is not required for the observed Ca 2ϩ -independent contraction or LC 20 diphosphorylation (17). In the case of Triton-skinned ileal smooth muscle strips, however, more recent studies have revealed contractile potentiation and possible activation of ZIPK upon the addition of the AV25 peptide (33). Unexpected variability in the effects of peptide inhibitors in situ, therefore, limits their usefulness in identifying substrates of ZIPK.…”

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

“…The pathway downstream of Rho kinase involves activation of ZIP kinase, a Ca 2ϩ -independent MLC kinase, and phosphorylation of the myosin phosphatase targeting subunit, MYPT1, resulting in inhibition of the catalytic subunit of MLC phosphatase. Phosphorylation of MLC 20 by ZIP kinase and inhibition of MLCP result in sustained muscle contraction (4,23,36). The pathway in circular smooth muscle, as previously shown, involves sequential activation of G q and/or G 13 , p115 RhoGEF, and RhoA (20,37,38,41,42).…”

“…The signaling pathway involves sequential activation of G␣ 13 , p115RhoGEF, RhoA/Rho kinase, and RhoA/PKC leading to inhibition of MLCP upon phosphorylation of its regulatory subunit, MYPT1 by Rho kinase, and phosphorylation of CPI-17, an endogenous inhibitor of MLCP, by PKC (4,41,42). ZIP kinase is activated downstream of Rho kinase and induces sustained MLC 20 phosphorylation and muscle contraction enhanced by a low ambient MLCP activity (23,36).In contrast, contraction of intestinal longitudinal smooth muscle is initiated by a distinct signaling pathway. Ca 2ϩ mobilization in longitudinal muscle is mediated by activation of cytosolic phospholipase A 2 (cPLA 2 ) and generation of arachidonic acid (AA), leading to Ca 2ϩ influx and stimulation of cyclic ADP ribose (cADPR) (30,31,39).…”

“…The signaling pathway involves sequential activation of G␣ 13 , p115RhoGEF, RhoA/Rho kinase, and RhoA/PKC leading to inhibition of MLCP upon phosphorylation of its regulatory subunit, MYPT1 by Rho kinase, and phosphorylation of CPI-17, an endogenous inhibitor of MLCP, by PKC (4,41,42). ZIP kinase is activated downstream of Rho kinase and induces sustained MLC 20 phosphorylation and muscle contraction enhanced by a low ambient MLCP activity (23,36).…”

Neonatal mouse ileum: functional properties and protein composition of the contractile machinery

“…Because P0 and adult ileum exhibit a similar dependence of force decline on MLC 20 dephosphorylation, relaxation may largely depend on MLCP activity without excluding the possibility that it is further rate limited by myosins with a slow rate of cross-bridge detachment such as NM myosin II (20,25) or slow SM myosin II isozymes, the latter being expressed in adult tonic SM (35) and possibly also in P0 ileum. The lower expression level of MLCK does not necessarily imply a lower MLCK/MLCP activity ratio as the specific activity of MLCK may be upregulated like in fetal carotid arteries (36) and a high Ca 2+ sensitivity may be supported by Ca 2+ independent MLC kinases (37). Their involvement in the P0 ileum remains to be determined.…”