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

Anne A. Wooldridge, Justin A. MacDonald, Ferenc Erdodi, Chaoyu Ma, Meredith A. Borman, David J. Hartshorne, Timothy A.J. Haystead

Research output: Contribution to journalArticlepeer-review

193 Scopus citations

Abstract

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca2+ sensitization/desensitization in smooth muscle. Ca2+ 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 Ca2+ 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 Ca2+ sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca2+ 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.

Original languageEnglish (US)
Pages (from-to)34496-34504
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number33
DOIs
StatePublished - Aug 13 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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