Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

2007 ◽  
Vol 292 (5) ◽  
pp. C1951-C1959 ◽  
Author(s):  
Eikichi Ihara ◽  
Elena Edwards ◽  
Meredith A. Borman ◽  
David P. Wilson ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Muscle Tension ◽  
Independent Manner ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca2+-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent Ki value of 3.4 μM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues.

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

2011 ◽  
Vol 286 (42) ◽  
pp. 36978-36991 ◽  
Author(s):  
Lori D. Moffat ◽  
Shannon B. A. Brown ◽  
Michael E. Grassie ◽  
Annegret Ulke-Lemée ◽  
Laura M. Williamson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Chemical Genetics ◽  
Arterial Smooth Muscle ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase ◽  
Bona Fide

The regulation of smooth muscle contractility by zipper-interacting protein kinaseThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

2007 ◽  
Vol 85 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Eikichi Ihara ◽  
Justin A. MacDonald
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Specific Inhibitor ◽  
Inhibitory Protein ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Independent Manner ◽  
Interacting Protein

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

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

2009 ◽  
Vol 297 (2) ◽  
pp. G361-G370 ◽  
Author(s):  
Eikichi Ihara ◽  
Lori Moffat ◽  
Meredith A. Borman ◽  
Jennifer E. Amon ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Conformational Changes ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

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 Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-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 Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 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 LC20 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.

Effect of calmodulin, Ca2+, and cAMP protein kinase on skinned tracheal smooth muscle

1984 ◽  
Vol 246 (3) ◽  
pp. C308-C314 ◽  
Author(s):  
M. P. Sparrow ◽  
G. Pfitzer ◽  
M. Gagelmann ◽  
J. C. Ruegg
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Progressive Increase ◽  
Response Curves ◽  
Tension Development ◽  
Regulation Of Contraction ◽  
Dependent Protein

The characteristics of contraction and relaxation of membrane skinned smooth muscle from guinea pig trachealis muscle are described. Micromolar Ca2+ elicited reproducible contractions in Mg-ATP salt solution at 20 degrees C. The speed of contraction was much faster at 30 and 37 degrees C, enabling cumulative concentration-response curves to be obtained. At these temperatures, a progressive increase in basal tension occurred in the absence of Ca2+. This tension was active and developed more rapidly at pH 6.7 than at pH 7.0. Calmodulin (0.1-10 microM) greatly increased the speed of contraction and lowered the threshold Ca2+ concentration ([Ca2+]) required to initiate contraction from 0.13 to 0.02 microM Ca2+. Trifluoperazine antagonized responses to Ca2+. Thiophosphorylation with adenosine 5'-O-(3-thiotriphosphate) produced maximum tension development, which was Ca2+-independent. This effect was reversible. The results are compatible with myosin-linked regulation of contraction in which a Ca2+ X calmodulin complex activates myosin light chain kinase to phosphorylate myosin. The catalytic subunit of cAMP-dependent protein kinase strongly inhibited tension development and slowly relaxed fibers contracted with threshold [Ca2+] consistent with an action via phosphorylation of myosin light chain kinase. This effect was extremely slow compared with the rate of relaxation by Ca2+ withdrawal or with relaxation of intact smooth muscle by beta-adrenergic agonists.

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