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 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. 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. Contraction and relaxation of smooth muscle are primarily determined by the level of phosphorylation of the myosin light chain (MLC-20). 1The abbreviations used are: MLC, myosin light chain; MLCK, MLC kinase; PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; ROCK, Rho kinase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GST, glutathione S-transferase; CRP, cleaved radioactive peptide; HPLC, high pressure liquid chromatography; ATPγS, adenosine 5′-O-(thiotriphosphate). To initiate contraction, an action potential or binding of a contractile agonist causes an increase in intracellular Ca2+, which activates myosin light chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme. MLCK phosphorylates MLC-20 on serine 19, resulting in contraction of smooth muscle through increases in myosin ATPase activity and cross-bridge cycling (1Horowitz A. Menice C.B. Laporte R. Morgan K.G. Physiol. Rev. 1996; 76: 967-1003Crossref PubMed Scopus (644) Google Scholar, 2Woodrum D.A. Brophy C.M. Mol. Cell. Endocrinol. 2001; 177: 135-143Crossref PubMed Scopus (32) Google Scholar). Smooth muscle myosin phosphatase (SMPP-1M) dephosphorylates MLC-20 resulting in relaxation of smooth muscle. Contraction can also occur in response to certain signals in the absence of changes in intracellular Ca2+, a phenomenon called Ca2+ sensitization. Inhibition of SMPP-1M activity is a primary mechanism of Ca2+ sensitization (3Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1686) Google Scholar, 4Seko T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (278) Google Scholar, 5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2002; 277: 23441-23446Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 6Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 7Ichikawa K. Ito M. Hartshorne D.J. J. Biol. Chem. 1996; 271: 4733-4740Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). One hypothesis is that SMPP-1M activity is inhibited by phosphorylation of the targeting subunit, MYPT1, at Thr-696 (of the human isoform). Several kinases have been shown to phosphorylate this site including Rho kinase (ROCK) (6Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), MYPT1-associated kinase (MYPT1K or ZIPK) (5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2002; 277: 23441-23446Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 8MacDonald J.A. Borman M.A. Muranyi A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google Scholar), myotonic dystrophy kinase (9Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar), and integrin-linked kinase (9Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar, 10Deng J.T. Van Lierop J.E. Sutherland C. Walsh M.P. J. Biol. Chem. 2001; 276: 16365-16373Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Relaxation of smooth muscle is achieved by either removal of the contractile agonist (passive relaxation) or through agonists that activate guanylyl or adenylyl cyclase and stimulate the production of cyclic nucleotides, cGMP or cAMP, thus activating their targets, cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) (2Woodrum D.A. Brophy C.M. Mol. Cell. Endocrinol. 2001; 177: 135-143Crossref PubMed Scopus (32) Google Scholar, 3Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1686) Google Scholar). These kinases lower intracellular Ca2+ through multiple mechanisms, but they also raise the Ca2+ threshold for contraction, thus causing Ca2+ desensitization (3Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1686) Google Scholar, 11Carvajal J.A. Germain A.M. Huidobro-Toro J.P. Weiner C.P. J. Cell. Physiol. 2000; 184: 409-420Crossref PubMed Scopus (314) Google Scholar). This is illustrated by the observation that GTPγS and carbachol-contracted smooth muscle relax in response to 8-bromo-cGMP at constant Ca2+ (12Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar). These findings suggest the mechanism of Ca2+ desensitization involves direct activation of SMPP-1M by the cyclic nucleotide-dependent kinases PKA and PKG. Indeed, several studies in permeabilized muscle have suggested that cGMP/PKG stimulates endogenous SMPP-1M activity; however, none have shown a direct activation (12Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar, 14Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. Chardin P. Pacaud P. Loirand G. J. Biol. Chem. 2000; 275: 21722-21729Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). In vitro the MYPT1 phosphorylation data suggest that multiple serine residues are phosphorylated by PKA/PKG; specifically Ser-849 (M133 isoform) was suggested because it is in a PKG phosphorylation consensus sequence, and the C-terminal portion of the protein was found to be phosphorylated (15Nakamura M. Ichikawa K. Ito M. Yamamori B. Okinaka T. Isaka N. Yoshida Y. Fujita S. Nakano T. Cell. Signal. 1999; 11: 671-676Crossref PubMed Scopus (52) Google Scholar). In smooth muscle, the presence of the leucine zipper domain of both PKG and MYPT1 has been shown to be essential for the protein kinase to mediate relaxation as determined by MLC-20 phosphorylation, although the binding of PKG-1 to MYPT1 does not absolutely require the leucine zipper domain (16Surks N. Y. Ito M. 1999; PubMed Scopus Google Scholar, Cell. Signal. 2003; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of smooth muscle with 8-bromo-cGMP with MYPT1 Thr-696 phosphorylation T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (278) Google Scholar). of SMPP-1M was but activity was by MLC suggested that phosphorylation by PKA/PKG Thr-696 phosphorylation and thus a in the level of inhibition of SMPP-1M an To the by which PKA/PKG Ca2+ desensitization through direct with the phosphorylated by kinases on MYPT1 in We serine residues on MYPT1 that are phosphorylated by PKA/PKG Ser-695, and human and showed specifically that phosphorylation of Ser-695 phosphorylation at the inactivating site Thr-696 by MYPT1K and in also of permeabilized smooth muscles with 8-bromo-cGMP induced phosphorylation of Ser-695 and the of MYPT1K. These findings suggest that cyclic mediate their in smooth muscle is by the phosphorylation mechanism for SMPP-1M. MYPT1 was by in by a recombinant of MYPT1 as the was in by the is This was by and MYPT1K MYPT1 and the serine to by cGMP-dependent protein kinase and the subunit of PKA and 8-bromo-cGMP was muscle was a and myosin A. K. P. C. Somlyo A.P. Somlyo A.V. Haystead T.A. J. Biol. Chem. Full Text PDF PubMed Google or MLC-20 (9Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google was as and of MYPT1 kinase the portion of the protein was as J.A. Borman M.A. Muranyi A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google Scholar). was in the in in at in for to protein and was by was cleaved by an with to the C-terminal of MYPT1 of the and and as (6Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, K. Hartshorne D.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Wu X. Haystead T.A. Somlyo A.P. P. Somlyo A.V. J. Biochem. 1996; PubMed Scopus Google Scholar). in contractile with and to smooth muscle was and of muscle and the with muscle in One of was to a and was to permeabilized with for at in with the Ca2+ for to intracellular Ca2+ in muscles with Ca2+ for and and in or 8-bromo-cGMP was for by for and in and of contraction as a of the contraction in of Smooth ileum was as and in a muscles permeabilized and as To the phosphorylation of Ser-695, the ileum was with or 8-bromo-cGMP for and in liquid To the level of Thr-696 phosphorylation with muscles with or 8-bromo-cGMP for by for and in liquid in in in and in and on a and to binding blocked with in with primary of MYPT1 or with primary MYPT1 and with for and with by and the phosphorylation was determined as a of the of protein was determined by of the In and phosphorylation for site by of MYPT1 of or PKG, and was phosphorylated by PKA or in the presence of and phosphatase was with an of Smooth muscle myosin phosphatase by myosin as A. K. P. C. Somlyo A.P. Somlyo A.V. Haystead T.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with the at with MYPT1 to and by on a in in and for phosphorylation of MYPT1 by PKA, ROCK, or as (9Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google by PKA or or was with and of the was and with a of was by as was by PKA for with as in A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google and and was to phosphorylation with in was phosphorylated by ROCK, as a of at to and also for in with the the of the the for inhibition of by as (9Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar). MYPT1 was either with or and by and to as J.A. Haystead T.A. Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in an to the immediately of the with of was determined by cleaved radioactive as the was used to residues in the MYPT1K that to of To the was with was as the of at with PKA/PKG phosphorylation on are as was used to the of Ser-695 and Thr-696 phosphorylation in smooth muscles and of changes in smooth muscle was of Smooth with the of of smooth muscles with 8-bromo-cGMP is to relaxation in a X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar). primary for cGMP is PKG, the by which the protein kinase Ca2+ desensitization are We to phosphorylation of MYPT1 by PKG the level of Ca2+ sensitization induced by MYPT1K. Addition of constitutively active to permeabilized smooth muscle is to phosphorylate MYPT1 endogenous SMPP-1M activity, and a Ca2+ sensitization (5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2002; 277: 23441-23446Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 8MacDonald J.A. Borman M.A. Muranyi A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google Scholar). effect of with 8-bromo-cGMP on contraction by was of permeabilized muscle with a contraction that was of the Ca2+ contraction and with 8-bromo-cGMP the contraction to of the Ca2+ contraction and findings suggest a mechanism of Ca2+ whereby phosphorylation of SMPP-1M or the of MYPT1K in smooth muscle. of the on by PKA/PKG in the hypothesis that their through phosphorylation of MYPT1, the phosphorylation by a recombinant of MYPT1 that PKG efficiently phosphorylated to of of that the phosphorylated protein was the in vitro phosphorylation was to To the phosphorylation was phosphorylated with PKA or PKG in the presence of and the phosphorylated protein was with C. was by HPLC, and in by of and to as J.A. Haystead T.A. Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in an and the of was determined by at at and and at and To the of the this of was on of with These to cycling was at as the at with Ser-692, Ser-695, and Ser-852 (of the human to and on the as phosphorylated by PKA/PKG on was the the Ser-852 and and of a of phosphorylation of showed that the phosphorylation of and Ser-695 was not site does not to be phosphorylated the can phosphorylation of in and was at PKA/PKG of MYPT1 SMPP-1M in effect of phosphorylation of MYPT1 by PKA/PKG on SMPP-1M activity was that been with the phosphatase subunit was phosphorylated by PKG. a with smooth muscle myosin as the of recombinant SMPP-1M by PKG does not phosphatase activity In contrast, phosphorylation of the by activity, which J.A. Borman M.A. Muranyi A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google that MYPT1K phosphorylates inhibiting SMPP-1M activity. for PKA, ROCK, and the in data suggested that of MYPT1 by PKA was the effect of of (M133 isoform) by PKA on the of SMPP-1M and to phosphorylate of by PKA to of of caused a in the phosphorylation of MYPT1 by and in to phosphorylation of MYPT1 by PKA and also in which of MYPT1 by phosphorylation by PKA that the of phosphorylation by PKA in the by is of a with of a with PKA with PKA and of by PKA phosphorylation by to the sequence, by of phosphorylation by PKA of of and blocked phosphorylation by These data suggest that the adjacent phosphorylation on MYPT1 for PKA, ROCK, and are on phosphorylation of the Although the in this is on the of the site Ser-695 and it is that the adjacent the site Ser-852 and be to the mutual to the of the adjacent Ser-695 and Thr-696 was to the was as a PKA/PKG the to was used to PKA and MYPT1K phosphorylated the to site that PKA phosphorylated Ser-695, phosphorylated Thr-696. To the of Ser-695 phosphorylation to with phosphorylation of the was in the presence and absence of PKA by was with and the was the of Thr-696 by was of of phosphorylation of the PKA was of of with PKA a in in the with sequence, by PKA, also blocked the of PKA to phosphorylate the at Ser-695 not In the in vitro (M133 isoform) was with PKA, and to phosphorylation by phosphorylation by PKA and in the of of by of by in the of of but in the of was These data suggested that both of and the the a level of phosphorylated Thr-696 in the to the of inhibition of by the at the of the shown in for phosphorylated by was with for the These data the level of phosphorylated Thr-696 in the with the in vitro data are with a potential in which phosphorylation of Ser-695 by PKA/PKG SMPP-1M activity by phosphorylation of the site by or In the phosphorylation by PKA a active for SMPP-1M. Ser-695 in Smooth in to and of This of Thr-696 SMPP-1M is by a mutual exclusion mechanism phosphorylation of Ser-695 in in smooth muscle, it was to a to this This was in the as the was a To the and of the recombinant MYPT1 was phosphorylated to with MYPT1K and PKA by was used to that Thr-696 and Ser-695 and the that the has a high of phosphorylated Ser-695 phosphorylated Thr-696 at a of that at the of MYPT1 the an of MYPT1, no with was the and of the Ser-695 Thr-696 at of MYPT1 Thr-696 phosphorylation by MYPT1K and ROCK, and is of PKA phosphorylated MYPT1 by this the of the in vitro, used the to the phosphorylation of Ser-695 in smooth muscle. the smooth muscle of muscle permeabilized in with (5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2002; 277: 23441-23446Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). intracellular of MYPT1 in smooth muscle has been determined to be and A. K. P. C. Somlyo A.P. Somlyo A.V. Haystead T.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). an intracellular of the of MYPT1 in smooth muscle is on of smooth muscle of MYPT1 to the phosphorylation of Ser-695 at of phosphorylation Ca2+ contraction, the muscles in and for with 8-bromo-cGMP or muscles in liquid and for with the Ser-695 is phosphorylated in smooth muscle at and with 8-bromo-cGMP increases the Ser-695 phosphorylation and To the of phosphorylation of Ser-695 and Thr-696 in smooth muscle, of ileum permeabilized with and with either or 8-bromo-cGMP for by for in liquid and for phosphorylation of Thr-696 and of muscles with 8-bromo-cGMP Thr-696 phosphorylation as with and that phosphorylation of Ser-695 phosphorylation of Thr-696 by MYPT1K the of shown in of the kinase to in this show that Ser-695 on MYPT1 is phosphorylated in smooth muscle in response to cGMP and phosphorylation of this site phosphorylation of Thr-696. These findings suggest a mechanism of Ca2+ desensitization mutual exclusion of of to phosphorylation of MYPT1, which prevents phosphorylation of Thr-696 by MYPT1K and ROCK, of SMPP-1M a mechanism of is with the level of of contraction that is of smooth muscle contractile of smooth muscle is by the of of MLCK and which the phosphorylation of or inhibition of either the kinase or phosphatase in on the level of phosphorylation of MLC-20 the contractile of the muscle. Regulation of SMPP-1M through phosphorylation of MYPT1 has been as a primary mechanism by which the contractile can be of Several protein kinases that specifically Thr-696 have been in to including ROCK, myotonic dystrophy and integrin-linked protein kinase (5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2002; 277: 23441-23446Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 6Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 8MacDonald J.A. Borman M.A. Muranyi A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google Scholar, A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar, 10Deng J.T. Van Lierop J.E. Sutherland C. Walsh M.P. J. Biol. Chem. 2001; 276: 16365-16373Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). of Thr-696 by kinases in smooth muscle has been shown to SMPP-1M and Ca2+ sensitization T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (278) Google Scholar, 5Borman M.A. MacDonald J.A. Muranyi A. Hartshorne D.J. Haystead T.A. J. Biol. 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Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (278) Google Scholar, N. S. J. Mol. 2002; PubMed Scopus Google Scholar). through G protein-coupled Ca2+ sensitization has been shown to be in multiple of and and RhoA or can be for of (3Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1686) Google Scholar, N. S. J. Mol. 2002; PubMed Scopus Google Scholar, Y. M. Res. 2001; PubMed Scopus Google Scholar, K. Y. H. A. T. K. T. T. M. J. 2000; PubMed Scopus Google Scholar, Y. H. H. M. J. 2001; PubMed Scopus Google Scholar, C. S. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). We have shown that 8-bromo-cGMP the of the for PKG to SMPP-1M in smooth muscle. Although MYPT1 is phosphorylated by PKA/PKG in smooth muscle, this does not SMPP-1M activity can activation of contractile activity through SMPP-1M In vitro data showed that phosphorylation of recombinant MYPT1 by PKA phosphorylation by and MYPT1K. also In vitro data also showed that of MYPT1 by PKA the of to phosphatase activity, an mechanism of phosphatase We found PKA/PKG phosphorylation on MYPT1 Ser-695, and We to Ser-695 because this site was adjacent to the Thr-696 hypothesis was that phosphorylation at this site SMPP-1M activity through mutual exclusion of mechanism to Ser-852 and but that was not in this level of in phosphorylation in vitro on recombinant MYPT1 suggested that both the Thr-696 and the on MYPT1 be by mutual exclusion of In vitro data and also showed that PKA phosphorylation the of MYPT1 phosphorylation by and Thr-696 phosphorylation is as Ser-695 phosphorylation Regulation of activity by mutual exclusion of phosphorylation is of this mechanism of occur with both and J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Biochem. 2003; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). on suggest that phosphorylation of Ser-695 in response to the of SMPP-1M activity through with acting through MYPT1K or In the mechanism in the of Ser-695 and Thr-696 phosphorylation as a to the of a mechanism of the smooth muscle contractile muscles to in a to a this mechanism is a is the of the in of the the of effect the activity of the phosphatase it be that the activity of the phosphatase for that for phosphatase activity for and to be We Ser-695 phosphorylation in In the permeabilized ileum smooth muscle, of this have been to in of the muscle. is also a level of phosphorylation, which the that Ca2+ desensitization is not an or mechanism of smooth muscle a of to which can to the (3Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1686) Google Scholar, 4Seko T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (278) Google Scholar, N. S. J. Mol. 2002; PubMed Scopus Google Scholar). Several are in the Ca2+ desensitization of smooth muscle induced by an increase in suggest but be in light of the data is the that phosphorylation of the site for of MYPT1 binding to myosin to SMPP-1M activity G. C. N. S. P. 2002; PubMed Scopus Google Scholar). in vitro that both be blocked by phosphorylation of adjacent PKA mechanism of MYPT1, on of phosphorylation (15Nakamura M. Ichikawa K. Ito M. Yamamori B. Okinaka T. Isaka N. Yoshida Y. Fujita S. Nakano T. Cell. Signal. 1999; 11: 671-676Crossref PubMed Scopus (52) Google Scholar, N. Y. Ito M. 1999; PubMed Scopus Google Scholar). to for in smooth muscle and K. H. Brophy C.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the in vitro have been and phosphorylation Ca2+ desensitization in permeabilized smooth muscle J.A. Somlyo A.V. Somlyo A.P. Haystead T.A. 2000; PubMed Scopus Google Scholar, S. K. M. J. Physiol. 2002; Scopus Google Scholar, X. Haystead T.A. Somlyo A.V. Somlyo A.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, MacDonald J.A. X. Haystead T.A. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (52) Google Scholar). it is that phosphorylation of Ser-695 in with to promote the relaxation of smooth muscle. of an the contractile in smooth In PKA/PKG phosphorylation on has no direct effect on SMPP-1M activity, the Ser-695 phosphorylation site as a potential mechanism of SMPP-1M activation through mutual exclusion of This that Ser-695 on MYPT1 is phosphorylated in smooth muscle in response to and phosphorylation of this site prevents phosphorylation of the site Thr-696. mechanism of Ca2+ desensitization is activation of to phosphorylation of Ser-695 of MYPT1, which prevents phosphorylation of Thr-696 by MYPT1K and of SMPP-1M. This mechanism of Ca2+ desensitization is a potential mechanism of smooth muscle which is essential for of We to H. for with

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