title: Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.
abstract: Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Anne A
authors.initials: AA
authors.lastname: Wooldridge
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
authors.forename: Ferenc
authors.initials: F
authors.lastname: Erdodi
authors.forename: Chaoyu
authors.initials: C
authors.lastname: Ma
authors.forename: Meredith A
authors.initials: MA
authors.lastname: Borman
authors.forename: David J
authors.initials: DJ
authors.lastname: Hartshorne
authors.forename: Timothy A J
authors.initials: TA
authors.lastname: Haystead
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D008954:Models, Biological; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry; D010766:Phosphorylation; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors
chemical_list: D051017:Apoptosis Regulatory Proteins; D009711:Nucleotides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013912:Threonine; C016276:8-bromocyclic GMP; D012694:Serine; D000242:Cyclic AMP; D064550:Death-Associated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D006152:Cyclic GMP; D002118:Calcium