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
2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês
10.1074/jbc.m405957200
ISSN1083-351X
AutoresAnne A. Wooldridge, Justin A. MacDonald, Ferenc Erdődi, Chaoyu Ma, Meredith A. Borman, David J. Hartshorne, Timothy Haystead,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoRegulation 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 H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (441) Google Scholar, 17Surks H.K. Mendelsohn M.E. Cell. Signal. 2003; 15: 937-944Crossref PubMed Scopus (45) Google Scholar, 18Huang Q.Q. Fisher S.A. Brozovich F.V. J. Biol. Chem. 2004; 279: 597-603Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Pretreatment of vascular smooth muscle cells with 8-bromo-cGMP before treatment with angiotensin II reduces MYPT1 Thr-696 phosphorylation (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). Activation of SMPP-1M was suspected, but activity was measured indirectly by assessing MLC dephosphorylation. The study suggested that phosphorylation by PKA/PKG may inhibit Thr-696 phosphorylation and thus a decrease in the level of inhibition of SMPP-1M rather than an activation. To investigate the mechanisms by which PKA/PKG might bring about Ca2+ desensitization through direct interactions with SMPP-1M, we first identified all the major sites phosphorylated by these kinases on MYPT1 in vitro. We identified three serine residues on MYPT1 that are phosphorylated by PKA/PKG (Ser-692, Ser-695, and Ser-852, human isoform numbering), and we showed specifically that phosphorylation of Ser-695 precludes phosphorylation at the inactivating site Thr-696 by MYPT1K and ROCK in vitro. The reverse situation also occurred. Treatment of permeabilized ileal smooth muscles with 8-bromo-cGMP induced phosphorylation of Ser-695 and greatly attenuated the Ca2+-sensitizing effects of exogenous MYPT1K. These findings suggest that one way cyclic nucleotides mediate their Ca2+-desensitizing effects in smooth muscle is by blocking the inhibitory phosphorylation mechanism for SMPP-1M. Materials—Affinity-purified MYPT1 antibody was made by Quality Control Biochemicals (Hopkington, MA) in rabbits by using a recombinant N-terminal fragment of rat MYPT1 as the antigen. Affinity-purified MYPT1pS695 antibody (Invitrogen) was produced in rabbits by using the peptide KLHRQSRRpSTQGVT (where pS is phosphoserine). This peptide was synthesized by Biomolecules Midwest (St. Louis, MO). Affinity-purified pMYPT1T696 antibody and ROCK (an N-terminal fragment) were from Upstate Group, Inc. (Charlottesville, VA). The MYPT1K substrate peptide from MYPT1 (689RQSRRSTQGVTL700) and the serine to alanine mutant peptide (689RQARRSTQGVTL700) (chicken M133 numbering) were synthesized by Biomolecules Midwest. cGMP-dependent protein kinase and the catalytic subunit of PKA were from Calbiochem, and 8-bromo-cGMP was from Sigma. Native PP-1c from rabbit skeletal muscle was a gift from Shirish Shenolikar (Durham, NC), and 32P-labeled myosin from pig bladder (19Shirazi A. Iizuka K. Fadden P. Mosse C. Somlyo A.P. Somlyo A.V. Haystead T.A. J. Biol. Chem. 1994; 269: 31598-31606Abstract Full Text PDF PubMed Google Scholar) or 32P-labeled gizzard 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 Scholar) was prepared as described. Expression and Purification of Recombinant Proteins—Recombinant MYPT1 kinase (rMYPT1K) encoding the N-terminal portion of the protein was produced as described from I.M.A.G.E. clone A1660136 (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). The cDNA clone was inserted in-frame into pGEX-6-P-1 in the NotI/BamHI sites (Amersham Biosciences). Escherichia coli cells were grown in LB broth in 50 μg/ml ampicillin overnight at 37 °C. Cells were incubated in 300 μm isopropyl-1-thio-β-d-galactopyranoside for 2 h to induce protein expression, and GST-MYPT1K was isolated by using glutathione-Sepharose. The GST moiety was cleaved by an overnight digestion with Precission Protease® according to the manufacturer's protocol (Amersham Biosciences). A hexahistidine-tagged C-terminal fragment of MYPT1 (sequence 514–963 of the M130 chicken isoform; termed C130), full-length chicken GST-MYPT1 (M133), and rat GST-MYPT1 (M110) were expressed and purified as described (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, 20Hirano K. Phan B.C. Hartshorne D.J. J. Biol. Chem. 1997; 272: 3683-3688Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Gailly P. Wu X. Haystead T.A. Somlyo A.P. Cohen P.T. Cohen P. Somlyo A.V. Eur. J. Biochem. 1996; 239: 326-332Crossref PubMed Scopus (44) Google Scholar). Muscle Tension Measurement—For in situ contractile experiments, adult male New Zealand White rabbits were anesthetized with halothane and exsanguinated according to approved animal protocols. Ileum smooth muscle was removed, and strips of muscle (5 mm × 200 μm) were cut and the ends tied with silk suture. The muscle strips were then mounted in 140-μl "bubble chambers." One end of each strip was attached to a force transducer (SensorOne AE801, Sausalito, CA), and tension was set to 1.3 times resting length. The strips were permeabilized with β-escin (50 μm) for 30 min at room temperature in Ca2+-free solution containing 1 mm EGTA (G1). Muscles were treated with the Ca2+ ionophore A23187 (10 μm) for 10 min to deplete intracellular Ca2+ stores. After extensive washing in G1, muscles were stimulated with 10 mm Ca2+ solution (CaG) for 5 min and then washed extensively and placed in G1. Either vehicle (water) or 8-bromo-cGMP (100 μm) was administered for 5 min, followed by rMYPT1K (10 μm) for 10 min. The strips were washed extensively again and then contracted in CaG. Force and rate of contraction were measured as a percent of the maximum contraction in CaG. Western Blot Analysis of Smooth Muscle Tissue—Rabbit ileum was collected as described above, and squares (5 × 5 mm) were mounted in a silicone bottom dish. The muscles were permeabilized and washed as described above. To test the phosphorylation of Ser-695, the ileum was treated with vehicle (water) or 8-bromo-cGMP (100 μm) for 10 min and then flash-frozen in liquid N2-cooled Freon. To test the level of Thr-696 phosphorylation after pretreatment with 8-bromo-cGMP, muscles were treated with vehicle or 8-bromo-cGMP (100 μm) for 5 min, followed by 10 μm rMYPT1K for 10 min, and then flash-frozen in liquid N2-cooled Freon. The frozen squares were stored in 10% trichloroacetic acid in acetone, thawed, washed in acetone, and then homogenized in buffer containing 600 mm NaCl, 25 mm Tris-HCl, pH 7.0, 0.5% Triton X-100, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm dithiothreitol, 2 mm EGTA, and 1 μm microcystin-LR. Homogenates were resolved on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.5% Tween 20. Blots were incubated overnight with primary antibody directed against phospho-Ser-695 of MYPT1 (1:1,000) or with primary polyclonal antibody directed against nonphosphorylated MYPT1 (1:2000). The blots were washed and incubated with horseradish peroxidase-conjugated rabbit secondary antibody (1:2000) for 1 h and developed with enhanced chemiluminescence (Amersham Biosciences). Bands were quantitated by densitometry, and the relative phosphorylation was determined as a function of the density of total MYPT1. Equal protein loading was determined by Amido Black staining of the polyvinylidene difluoride membranes after blotting. In Vitro Phosphorylation, Kinase, and Phosphatase Assays—All phosphorylation reactions for site analysis were carried out by using 100 μg of MYPT1 (c130), 5 μg of PKAc or PKG, 50 mm HEPES, pH 7.2, 1 mm MgCl2, and 0.2 mm [γ-32P]ATP (2,500 cpm/nmol). For stoichiometry reactions, 2 μm GST-MYPT1 (or C130) was phosphorylated by PKA (1 μg/ml), ROCK (0.4 units/ml), or rMYPT1K (44 μg/ml) in the presence of 50 mm HEPES, pH 7.2, 1 mm MgCl2, and 0.2 mm [γ-32P]ATP (2500 cpm/nmol). For phosphatase assays, GST-MYPT1 (M110) was mixed with an equimolar amount of native rabbit PP1C. Smooth muscle myosin phosphatase assays were performed by using 32P-labeled myosin as described previously (19Shirazi A. Iizuka K. Fadden P. Mosse C. Somlyo A.P. Somlyo A.V. Haystead T.A. J. Biol. Chem. 1994; 269: 31598-31606Abstract Full Text PDF PubMed Google Scholar). Kinase assays with the substrate peptide were carried out at 25 °Cin50-μl reactions with 50 mm HEPES, pH 7.2, 1 mm MgCl2, 0.2 mm [γ-32P]ATP (2500 cpm/nmol), 100 μm MYPT1 Ser-691 to Ala substrate peptide, and 0.3 μm rMYPT1K. Reactions were terminated by spotting on P81 paper after a time course. P81 papers were washed three times in 20 mm H3PO4, placed in 1.5-ml Eppendorf tubes, and Cerenkov counted. Kinase assays for sequential phosphorylation of MYPT1 by PKA, ROCK, or rMYPT1K were carried out as described previously (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) by using GST-MYPT1 (2 μm), PKA (1 μg/ml), or ROCK (0.4 units/ml) or rMYPT1K (44 μg/ml). Phosphorylation was initiated with one kinase, and then part of the reaction mixture was removed and continued with a second kinase. Incorporation of 32P was estimated by Cerenkov counting as above. GST-MYPT1 (2 μm) was thiophosphorylated by PKA (1 μg/ml) for 150 min with 0.5 mm ATPγS (other conditions as in Ref. 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 dialyzed against 20 mm Tris-HCl, pH 7.5, 85 mm KCl, 5 mm MgCl2, 10 mm EGTA, and 4 mm dithiothreitol. The thiophosphorylated GST-MYPT1 was then subjected to phosphorylation with ROCK (0.4 units/ml) in 0.2 mm [γ-32P]ATP. A non-thiophosphorylated GST-MYPT1 was phosphorylated by ROCK, as a control. Aliquots of each reaction were removed at intervals to measure phosphate incorporation and also for use in Western blots with the MYPT1pT696 antibody. At the end of the reaction period, the two samples were assayed for inhibition of native PP1c (0.5 nm) by using 32P-MLC-20 (5 μm) as substrate (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). Phosphorylation Site Analysis—Phosphorylated MYPT1 was digested either with endoprotease Lys-C (6 μg/mg) or V8 endoprotease E (V8 protease) (6 μg/mg) overnight, and phosphopeptides were separated by reverse phase HPLC. Radioactive peptides were collected and cross-linked to Immobilon P filters (Applied Biosystems) as described (22MacDonald J.A. Mackey A.J. Pearson W.R. Haystead T.A. Mol. Cell Proteomics. 2002; 1: 314-322Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The filters were placed in an ABI 474cLC Edman sequenator configured to collect the phenylthiohydantoin-derivatives immediately following cleavage of the N-terminal amino acid with trifluoroacetic acid. The amount of radioactivity released after each Edman cycle was determined by Cerenkov counting. The cleaved radioactive peptide (CRP) algorithm (fasta.bioch.virginia.edu/crp/) using lysine as the cleavage point was used to identify all possible serine/threonine residues in the MYPT1K sequence that corresponded to release of radioactivity during Edman sequencing. To precisely identify each site, the experiment was repeated following digestion with V8 protease. CRP analysis was repeated using glutamic acid as the cleavage site. Cross-referencing release of radioactivity after cleavage at lysine with glutamic acid unambiguously identified three PKA/PKG phosphorylation sites on MYPT1. Statistical Analysis—All results are reported as mean ± S.E. A Student's unpaired t test was used to determine the statistical significance of Ser-695 and Thr-696 phosphorylation in smooth muscles and of changes in smooth muscle contraction. p < 0.05 was considered significant. Treatment of Smooth Muscle Strips with 8-Bromo-cGMP Attenuates the Ca2+-sensitizing Effect of MYPT1K—Treatment of isolated smooth muscles with 8-bromo-cGMP is known to bring about relaxation in a Ca2+-independent manner (13Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar). The primary target for cGMP is PKG, yet the mechanisms by which the protein kinase brings about Ca2+ desensitization are unclear. We sought to determine whether phosphorylation of MYPT1 by PKG reduced the level of Ca2+ sensitization induced by MYPT1K. Addition of constitutively active rMYPT1K to permeabilized smooth muscle is known to phosphorylate MYPT1 (at Thr-696), inhibit endogenous SMPP-1M activity, and produce a marked 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). The effect of pretreatment with 8-bromo-cGMP on contraction elicited by rMYPT1K was analyzed. Treatment of permeabilized muscle with rMYPT1K elicited a contraction that was 41.8 ± 0.9% of the maximum Ca2+ (CaG) contraction (Fig. 1, A and C). Pre-treatment with 100 μm 8-bromo-cGMP before rMYPT1K administration significantly reduced the contraction to 19 ± 6.54% (n = 5, p < 0.04) of the maximum Ca2+ contraction (Fig. 1, B and C). The findings suggest a possible mechanism of Ca2+ desensitization, whereby phosphorylation of SMPP-1M or other proteins attenuates the Ca2+-sensitizing actions of MYPT1K in smooth muscle. Characterization of the Site Phosphorylated on rMYPT1 by PKA/PKG in Vitro—To investigate the hypothesis that PKG/PKA may bring about their Ca2+-desensitizing effects through phosphorylation of MYPT1, we identified the phosphorylation sites by using a recombinant fragment of MYPT1 (C130). Fig. 2A (inset) shows that PKG efficiently phosphorylated C130 to ∼3.5 mol of phosphate/mol of C130. Phosphoamino acid analysis revealed that the fully phosphorylated protein contained phosphoserine. Some phosphothreonine was seen if the in vitro phosphorylation reaction was carried out to 2.5 h. To identify the phosphorylation sites, C130 was phosphorylated with PKA or PKG in the presence of [γ-32P]ATP, and the phosphorylated protein was digested with endolysine protease C. The digest was separated by reverse phase HPLC, and phosphopeptides were identified in column fractions by Cerenkov counting (Fig. 2A). Three peaks of radioactivity were identified and cross-linked to Immobilon P filters (Applied Biosystems, Inc.) as described (22MacDonald J.A. Mackey A.J. Pearson W.R. Haystead T.A. Mol. Cell Proteomics. 2002; 1: 314-322Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The Immobilon filters were placed in an automated Edman sequenator, and after each Edman cycle, the amount of radioactivity released from each cross-linked peptide was determined by Cerenkov counting (Fig. 2, B–D). Peak 1 yielded radioactivity at cycle 3; peak 2 at cycles 4, 9, and 12; and peak 3 at cycles 4 and 12. To confirm the identity of the sites, this cycle of analysis was repeated on phosphopeptides recovered from reverse phase HPLC separation after digestion of PKA/PKG-phosphorylated C130 with V8 endoprotease E (Fig. 2E). These three peptides were subjected to Edman cycling (Fig. 2 F–H). CRP analysis was then carried out using cleavage at glutamic acid as the reference point. Cross-referencing CRP analysis at lysine with glutamic acid unambiguously identified Ser-692, Ser-695, and Ser-852 (of the human isoform, corresponding to Ser-650, Ser-653, and Ser-808 on the chicken M130 protein) as sites phosphorylated by PKA/PKG on rMYPT1. Peak I (endoprotease Lys-C) was the peptide containing the Ser-852 site, and peak II (endoprotease Lys-C) contained Ser-692 and Ser-695. Analysis of a time course of phosphorylation of peptide II showed that the phosphorylation of Ser-692 and Ser-695 was not ordered, i.e. one site does not need to be phosphorylated before the other one can be. Some phosphorylation of threonine in peak 2 (endolysine protease C) and peak 3 (V8 E) was noted at later time points. PKA/PKG Phosphorylation of MYPT1 Does Not Affect SMPP-1M Activity in Vitro—The effect of phosphorylation of MYPT1 by PKA/PKG on SMPP-1M activity was determined. Purified full-length rMYPT1 (M110) that had been incubated with the phosphatase catalytic subunit PP-1C was phosphorylated by PKG. As a control, parallel reactions using rMYPT1K were performed. Phosphatase assays were carried out with 32P-labeled smooth muscle myosin as the substrate. Phosphorylation of recombinant SMPP-1M by PKG does not affect phosphatase activity (Fig. 3A). In contrast, phosphorylation of the enzyme by rMYPT1K dramatically inhibits activity, which corroborates previous work (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) showing that MYPT1K preferentially phosphorylates Thr-696, inhibiting SMPP-1M activity. The Phosphorylation Sites for PKA, ROCK, and rMYPT1K Are Mutually Dependent—Because the in situ data suggested that pre-phosphorylation of MYPT1 by PKA was affecting calcium sensitization, we investigated the effect of pre-phosphorylation of GST-MYPT1 (M133 isoform) by PKA on the ability of known SMPP-1M inhibitory kinases, ROCK and MYPT1K, to phosphorylate rMYPT1. Pre-phosphorylation of GST-MYPT1 (M133) by PKA to 2 mol of phosphate/mol of GST-MYPT1 caused a dramatic decrease in the subsequent phosphorylation of MYPT1 by ROCK and 0.26 mol/mol in contrast to 1.60 mol/mol phosphorylation of MYPT1 by ROCK without PKA pre-phosphorylation (Fig. 3, B and C). The reverse reaction also occurs in which pre-phosphorylation of MYPT1 by ROCK decreased further phosphorylation by PKA (Fig. 3C). Note that the extent of phosphorylation by PKA in the sample pre-phosphorylated by ROCK is about 0.95 mol of phosphate/mol (over a 150-min incubation) compared with about 2.2 mol of phosphate/mol (over a similar time period) with PKA alone (Fig. 3B). Similar experiments were carried out with PKA and rMYPT1K. Pre-phosphorylation of GST-MYPT1K by PKA reduced subsequent phosphorylation by rMYPT1K to about 0.4 mol phosphate/mol (Fig. 3D). By using the opposite sequence, pre-phosphorylation by rMYPT1K (to about 1.7 mol of phosphate/mol) attenuated phosphorylation by PKA (increase of only 0.25 mol of phosphate/mol) and essentially blocked phosphorylation by ROCK (Fig. 3E). These data suggest that the adjacent phosphorylation sites on MYPT1 for PKA, ROCK, and rMYPT1K are dependent on phosphorylation of the partner site. Although the emphasis in this article is on the pair of sites containing the inhibitory site (i.e. Ser-695 and Thr-696), it is evident that the adjacent sites containing the second major ROCK site (i.e. Ser-852 and Thr-853) may be subject to the same mutual dependence. Another approach to assess the influence of the adjacent Ser-695 and Thr-696 sites was to use the synthetic substrate peptide (residues 689–700). Because Ser-691 (avian numbering) was identified as a PKA/PKG site, the Ser-691 to Ala mutant was used (see "Experimental Procedures") to simplify analysis. Both PKA and MYPT1K readily phosphorylated the peptide to 1 mol/mol. Phosphorylation site analysis using radioactivity release during Edman sequence analysis confirmed that PKA phosphorylated Ser-695, whereas rMYPT1K phosphorylated Thr-696. To test the ability of Ser-695 phosphorylation to interfere with phosphorylation of Thr-696, the peptide was pre-phosphorylated in the presence and absence of PKA by using nonradioactive ATP. After 30 min, rMYPT1K was added with [γ-32P]ATP, and the reaction wa
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