Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide.
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 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.
Ihara
Eikichi
E
Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada.
Moffat
Lori
L
Borman
Meredith A
MA
Amon
Jennifer E
JE
Walsh
Michael P
MP
MacDonald
Justin A
JA
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002645:Chickens; D064550:Death-Associated Protein Kinases; D004789:Enzyme Activation; D005769:Gastrointestinal Motility / Q000187:drug effects*; D007082:Ileum / Q000187:drug effects* / Q000201:enzymology; D066298:In Vitro Techniques; D052998:Microcystins / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects*; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D009154:Mutation; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1 / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D013379:Substrate Specificity
D051017:Apoptosis Regulatory Proteins; D052998:Microcystins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010446:Peptide Fragments; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D047428:Protein Kinase Inhibitors; C090954:SM 1 peptide; C078588:microcystin; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D002118:Calcium