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, F. Erdődi, Chaoyu Ma, Meredith A. Borman, David J. Hartshorne, Timothy A J Haystead

Research output: Contribution to journalArticle

169 Citations (Scopus)

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
Pages (from-to)34496-34504
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number33
DOIs
Publication statusPublished - Aug 13 2004

Fingerprint

Phosphorylation
Cyclic Nucleotides
Threonine
Phosphoric Monoester Hydrolases
Serine
Smooth Muscle
Muscle
Protein Kinases
Protein Isoforms
Chemical activation
Myosin-Light-Chain Phosphatase
Smooth Muscle Myosins
Cyclic GMP-Dependent Protein Kinases
Myosins
G-Protein-Coupled Receptors
Cyclic AMP-Dependent Protein Kinases
Ileum

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Wooldridge, Anne A.; MacDonald, Justin A.; Erdődi, F.; Ma, Chaoyu; Borman, Meredith A.; Hartshorne, David J.; Haystead, Timothy A J.

In: Journal of Biological Chemistry, Vol. 279, No. 33, 13.08.2004, p. 34496-34504.

Research output: Contribution to journalArticle

Wooldridge, Anne A. ; MacDonald, Justin A. ; Erdődi, F. ; Ma, Chaoyu ; Borman, Meredith A. ; Hartshorne, David J. ; Haystead, Timothy A J. / 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. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 33. pp. 34496-34504.
@article{b040fe7a96224eef8ac38004963448a7,
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 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.",
author = "Wooldridge, {Anne A.} and MacDonald, {Justin A.} and F. Erdődi and Chaoyu Ma and Borman, {Meredith A.} and Hartshorne, {David J.} and Haystead, {Timothy A J}",
year = "2004",
month = "8",
day = "13",
doi = "10.1074/jbc.M405957200",
language = "English",
volume = "279",
pages = "34496--34504",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "33",

}

TY - JOUR

T1 - 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

AU - Wooldridge, Anne A.

AU - MacDonald, Justin A.

AU - Erdődi, F.

AU - Ma, Chaoyu

AU - Borman, Meredith A.

AU - Hartshorne, David J.

AU - Haystead, Timothy A J

PY - 2004/8/13

Y1 - 2004/8/13

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=4544321243&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4544321243&partnerID=8YFLogxK

U2 - 10.1074/jbc.M405957200

DO - 10.1074/jbc.M405957200

M3 - Article

C2 - 15194681

AN - SCOPUS:4544321243

VL - 279

SP - 34496

EP - 34504

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 33

ER -