title: Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase.
abstract: Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.
authors.affiliation: Smooth Muscle Research Group and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta T2N 4Z6, Canada.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Grassie
authors.forename: Cindy
authors.initials: C
authors.lastname: Sutherland
authors.forename: Annegret
authors.initials: A
authors.lastname: Ulke-Lemée
authors.forename: Mona
authors.initials: M
authors.lastname: Chappellaz
authors.forename: Enikö
authors.initials: E
authors.lastname: Kiss
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D020125:Mutation, Missense; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D024745:Smooth Muscle Myosins / Q000235:genetics / Q000378:metabolism*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D014662:Vasoconstrictor Agents; D005576:Colforsin; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000242:Cyclic AMP; D054460:rho-Associated Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D024745:Smooth Muscle Myosins; D020742:rhoA GTP-Binding Protein