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      Regulation of gastric smooth muscle contraction via Ca 2+-dependent and Ca 2+-independent actin polymerization

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          Abstract

          In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser 19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca 2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca 2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gα q, IP 3 formation, Ca 2+ release and Ca 2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr 402. The sustained Ca 2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr 397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr 118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC 20 phosphorylation.

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          Most cited references56

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          Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.

          Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes. We conclude that the majority of phenotypes attributed to the monomeric G protein RhoA and mediated by its effector, Rho-kinase (ROK), reflect Ca2+ sensitization: inhibition of myosin II dephosphorylation in the presence of basal (Ca2+ dependent or independent) or increased MLCK activity. We outline the pathway from receptors through trimeric G proteins (Galphaq, Galpha12, Galpha13) to activation, by guanine nucleotide exchange factors (GEFs), from GDP. RhoA. GDI to GTP. RhoA and hence to ROK through a mechanism involving association of GEF, RhoA, and ROK in multimolecular complexes at the lipid cell membrane. Specific domains of GEFs interact with trimeric G proteins, and some GEFs are activated by Tyr kinases whose inhibition can inhibit Rho signaling. Inhibition of MLCP, directly by ROK or by phosphorylation of the phosphatase inhibitor CPI-17, increases phosphorylation of the myosin II regulatory light chain and thus the activity of smooth muscle and nonmuscle actomyosin ATPase and motility. We summarize relevant effects of p21-activated kinase, LIM-kinase, and focal adhesion kinase. Mechanisms of Ca2+ desensitization are outlined with emphasis on the antagonism between cGMP-activated kinase and the RhoA/ROK pathway. We suggest that the RhoA/ROK pathway is constitutively active in a number of organs under physiological conditions; its aberrations play major roles in several disease states, particularly impacting on Ca2+ sensitization of smooth muscle in hypertension and possibly asthma and on cancer neoangiogenesis and cancer progression. It is a potentially important therapeutic target and a subject for translational research.
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            The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.

            Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. We describe a novel mechanism that links one Rho family member, Cdc42, to actin polymerization. N-WASP, a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization.
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              Paxillin: a focal adhesion-associated adaptor protein.

              Paxillin is a focal adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the focal adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Methodology
                Role: Data curationRole: Methodology
                Role: MethodologyRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: ResourcesRole: SoftwareRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                20 December 2018
                2018
                : 13
                : 12
                : e0209359
                Affiliations
                [001]Department of Physiology and Biophysics, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, Virginia, United States of America
                Institute of Biochemistry and Cell Biology, SINGAPORE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-9760-4585
                http://orcid.org/0000-0001-6567-5023
                Article
                PONE-D-18-19424
                10.1371/journal.pone.0209359
                6301582
                30571746
                f0529103-acb6-49ee-a93a-b23e9c0fd17d
                © 2018 Mahavadi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 June 2018
                : 4 December 2018
                Page count
                Figures: 16, Tables: 1, Pages: 34
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases;
                Award ID: DK28300
                Award Recipient : Karnam S Murthy
                This work was supported by National Institute of Diabetes and Digestive Kidney Diseases Grant to K. S. Murthy (DK 28300), https://www.niddk.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Actin Polymerization
                Biology and Life Sciences
                Physiology
                Muscle Physiology
                Muscle Contraction
                Medicine and Health Sciences
                Physiology
                Muscle Physiology
                Muscle Contraction
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Muscle Cells
                Smooth Muscle Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Muscle Tissue
                Muscle Cells
                Smooth Muscle Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Muscle Tissue
                Muscle Cells
                Smooth Muscle Cells
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Phosphorylation
                Biology and Life Sciences
                Anatomy
                Musculoskeletal System
                Muscles
                Smooth Muscles
                Medicine and Health Sciences
                Anatomy
                Musculoskeletal System
                Muscles
                Smooth Muscles
                Research and Analysis Methods
                Precipitation Techniques
                Immunoprecipitation
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzyme Inhibitors
                Kinase Inhibitors
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Muscle Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Muscle Tissue
                Muscle Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Muscle Tissue
                Muscle Cells
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                All relevant data are within the paper.

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