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.
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.
Wooldridge
Anne A
AA
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
MacDonald
Justin A
JA
Erdodi
Ferenc
F
Ma
Chaoyu
C
Borman
Meredith A
MA
Hartshorne
David J
DJ
Haystead
Timothy A J
TA
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
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