Molecular Determinants of the Interaction between the Inositol 1,4,5-Trisphosphate Receptor-associated cGMP Kinase Substrate (IRAG) and cGMP Kinase Iβ

Abstract

Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP3)-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate). IRAG is present in a macromolecular complex with the InsP3 receptor type I (InsP3RI) and cGKIβ. The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins. The amino-terminal region containing the leucine zipper (amino acids 1–53) of cGKIβ but not that of cGKIα or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitroand in vivo most likely through electrostatic interaction. cGKIβ did not interact with the InsP3RI, but co-precipitated the InsP3RI in the presence of IRAG indicating that IRAG bound to the InsP3RI and to cGKIβ. cGKIβ phosphorylated up to four serines in IRAG. Mutation of these four serines to alanine showed that cGKIβ-dependent phosphorylation of Ser696 is necessary to decrease calcium release from InsP3-sensitive stores. These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIβ and phosphorylation of Ser696 of IRAG. Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP3)-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate). IRAG is present in a macromolecular complex with the InsP3 receptor type I (InsP3RI) and cGKIβ. The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins. The amino-terminal region containing the leucine zipper (amino acids 1–53) of cGKIβ but not that of cGKIα or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitroand in vivo most likely through electrostatic interaction. cGKIβ did not interact with the InsP3RI, but co-precipitated the InsP3RI in the presence of IRAG indicating that IRAG bound to the InsP3RI and to cGKIβ. cGKIβ phosphorylated up to four serines in IRAG. Mutation of these four serines to alanine showed that cGKIβ-dependent phosphorylation of Ser696 is necessary to decrease calcium release from InsP3-sensitive stores. These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIβ and phosphorylation of Ser696 of IRAG. cyclic GMP-dependent protein kinase cyclic GMP-dependent protein kinase type I or II inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate inositol 1,4,5-trisphosphate receptor type I inositol 1,4,5-trisphosphate polymerase chain reaction 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis Signal transduction via NO/cGMP/cGKI1 is involved in a variety of cellular mechanisms including smooth muscle contractility and platelet aggregation (1Francis S.H. Corbin J.D. Adv. Pharmacol. 1994; 26: 115-170Crossref PubMed Scopus (77) Google Scholar, 2Lincoln T.M. Komalavilas P. Cornwell T.L. Hypertension. 1994; 23: 1141-1147Crossref PubMed Scopus (173) Google Scholar, 3Pfeifer A. Ruth P. Dostmann W. Sausbier M. Klatt P. Hofmann F. Rev. Physiol. Biochem. Pharmacol. 1999; 135: 105-149Crossref PubMed Google Scholar, 4Hofmann F. Ammendola A. Schlossmann J. J. Cell Sci. 2000; 113: 1671-1676Crossref PubMed Google Scholar). cGKI affects smooth muscle tone by either decreasing the release of calcium from InsP3-sensitive stores (5Felbel J. Trockur B. Ecker T. Landgraf W. Hofmann F. J. Biol. Chem. 1988; 263: 16764-16771Abstract Full Text PDF PubMed Google Scholar, 6Francis S.H. Noblett B.D. Todd B.W. Wells J.N. Corbin J.D. Mol. Pharmacol. 1988; 34: 506-517PubMed Google Scholar, 7Lincoln T.M. Cornwell L.T. Taylor A.E. Am. J. Physiol. 1990; 258: C399-C407Crossref PubMed Google Scholar, 8Pfeifer A. Klatt P. Massberg S. Ny L. Sausbier M. Hirneiß C. Wang G. Korth M. Aszódi A. Andersson E. Krombach F. Mayerhofer A. Ruth P. Fässler R. Hofmann F. EMBO J. 1998; 17: 3045-3051Crossref PubMed Scopus (459) Google Scholar, 9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar) or by reducing calcium sensitivity of the contractile elements (10Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 11Somlyo A.P. Somlyo A.V. J. Physiol. ( Lond .). 2000; 522: 177-185Crossref PubMed Scopus (1085) Google Scholar). During the last years, several mechanisms were proposed for the action of cGKI mediating these effects. A decrease of the cytosolic calcium concentration by cGKI might involve reduced InsP3 synthesis (12Ruth P. Wang G.X. Boekhoff I. May B. Pfeifer A. Penner R. Korth M. Breer H. Hofmann F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2623-2627Crossref PubMed Scopus (107) Google Scholar, 13Pfeifer A. Nürnberg B. Kamm S. Uhde M. Schultz G. Ruth P. Hofmann F. J. Biol. Chem. 1995; 270: 9052-9059Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 14Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1998; 273: 34519-34526Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Wang G.R. Zhu Y. Halushka P.V. Lincoln T.M. Mendelsohn M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4888-4893Crossref PubMed Scopus (232) Google Scholar), enhanced calcium re-uptake by intracellular stores via CaATPase (16Cohen R.A. Weisbrod R.M. Gericke M. Yaghoubi M. Bierl C. Bolotina V.M. Circ. Res. 1999; 84: 210-219Crossref PubMed Scopus (260) Google Scholar), or inhibition of calcium release via the InsP3R (17Tertyshnikova S. Yan X. Fein A. J. Physiol. ( Lond .). 1998; 512.1: 89-96Crossref Scopus (54) Google Scholar). The molecular mechanisms for these different possible intracellular calcium regulation pathways were only partly resolved up to now. Recently, we identified a 125-kDa cGKI substrate protein which was designated as inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate (IRAG). IRAG which is phosphorylated by cGKI is associated in a macromolecular complex with cGKIβ and InsP3RI in smooth muscle (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). The observed perinuclear localization of heterologously expressed IRAG suggested the potential role of IRAG as a modulator of calcium release from intracellular stores. Indeed, functional studies revealed that IRAG inhibits InsP3-induced calcium release after activation of cGKIβ with 8-pCPT-cGMP in COS-7 cells (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). However, the precise mechanism by which IRAG influences calcium release is still unknown. In the present study we investigated the molecular determinants for the interaction of IRAG and cGKI. It is shown that IRAG interacts specifically with the amino-terminal region containing the leucine zipper of cGKIβ. Phosphorylation of Ser696 of IRAG is essential for the inhibition of InsP3-induced calcium release. The yeast strain EGY48 (MATα, his3, trp1, ura3, lexAopx6 Leu2) and the yeast expression plasmids pEG202, pJG4-5, and pSH18-34 were used for the two-hybrid screen. Yeast media and drop-out media lacking the appropriate amino acids were obtained from CLONTECH (Heidelberg, Germany) and Difco (Hamburg, Germany), respectively. The full-length rat cDNAs of the neuronal InsP3RI (S1−/S2+) (18Mignery G.A. Newton C.L. Archer B.T. Südhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar) and the peripheral InsP3RI (S1−/S2−) (19Kaznacheyeva E. Lupu V.D. Bezprozvanny I. J. Gen. Physiol. 1998; 111: 847-856Crossref PubMed Scopus (62) Google Scholar) were a gift from Dr. Ilya Bezprozvanny (Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX). A polyclonal antibody specific for IRAG, raised in rabbits against recombinant IRAG53–499 expressed in bacteria, was used for Western blot analysis at a dilution of 1:1000. Further antibodies were directed against cGKI (8Pfeifer A. Klatt P. Massberg S. Ny L. Sausbier M. Hirneiß C. Wang G. Korth M. Aszódi A. Andersson E. Krombach F. Mayerhofer A. Ruth P. Fässler R. Hofmann F. EMBO J. 1998; 17: 3045-3051Crossref PubMed Scopus (459) Google Scholar) and InsP3RI (ABR Biochemicals). The bovine IRAG cDNA was the template of all IRAG constructs (baits) which were synthesized by PCR. The generated IRAG amplicons were purified and inserted into the BamHI/EcoRI-digested expression plasmid pEG202 (20Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Current Protocols in Molecular Biology, pp. 13.1.1–13.1.6 and 20.1.1–20.1.28, John Wiley & Sons, Inc., New York.Google Scholar) in-frame with the DNA-binding domain of LexA, yielding pEG202-LexA/IRAG. The full-length and truncated PCR amplicons of bovine cGKIα and cGKIβ were ligated into theEcoRI/XhoI sites of the pJG4-5 expression plasmid (20Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Current Protocols in Molecular Biology, pp. 13.1.1–13.1.6 and 20.1.1–20.1.28, John Wiley & Sons, Inc., New York.Google Scholar) and used as preys. The inserts were fused in-frame to the “acid loop” DNA activation domain of pJG4-5, yielding pJG4-5/cGKI. As cGKII Prkg2 gene from mouse contains an internalXhoI site it was subcloned as EcoRI fragment into pJG4-5. The correct orientation was checked by sequence analysis. The full-length rat InsP3RI (S1−/S2−) without the channel forming domain was divided into five different fragments. These inserts were amplified by PCR and ligated into pJG4-5 via EcoRI andXhoI in order to obtain various pJG4-5/InsP3RI constructs. cGKIβ was generated as bait throughEcoRI/XhoI digestion of pJG4-5/cGKIβ and subsequent cloning of the isolated cGKIβ fragment into pEG202 digested with the same enzymes. Recombinant plasmids were transformed in Escherichia coli XL1-Blue. The correct reading frame was verified by sequence analysis. All used primer pairs are listed at the supplement. Growth, maintenance, and screening of the yeast cells has been performed as described (20Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Current Protocols in Molecular Biology, pp. 13.1.1–13.1.6 and 20.1.1–20.1.28, John Wiley & Sons, Inc., New York.Google Scholar). The plasmid pSH18-34 (20Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Current Protocols in Molecular Biology, pp. 13.1.1–13.1.6 and 20.1.1–20.1.28, John Wiley & Sons, Inc., New York.Google Scholar), the different and plasmids were by R.A. 1995; PubMed Scopus Google Scholar) in the yeast strain The expression of IRAG as protein was by of cells appropriate constructs by and analysis with antibodies directed against IRAG. were lacking and As protein interaction sites were investigated in the performed was with the described by F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Current Protocols in Molecular Biology, pp. 13.1.1–13.1.6 and 20.1.1–20.1.28, John Wiley & Sons, Inc., New York.Google Scholar). results in activation of the were identified by and to media lacking leucine in the presence of of IRAG phosphorylation sites from to alanine was performed by PCR to H.D. R.M. ( .). PubMed Scopus Google Scholar). the of the and a DNA fragment primer are In a PCR DNA were generated which were used as template in a PCR with the primer The primer pairs used for the of phosphorylation sites and the for the of the cDNA are listed in of the supplement. of was performed using plasmids as template IRAG amplicons were purified and ligated into after digestion with the same and used for the in phosphorylation of used for calcium PCR were into All were by sequence analysis COS-7 cells in were with IRAG constructs into phosphorylation or calcium by using the calcium of at cells were for or used for calcium respectively. COS-7 cells with the different cGKIβ in or InsP3RI in were with and in using a were at with and in were using a with a and in at from COS-7 cells were for IRAG, and InsP3RI using analysis with specific Cell containing of IRAG and cGKIβ were used for phosphorylation of were in in the presence or of 8-pCPT-cGMP for up to at The were by of were by and to was by and analysis of was performed using the analysis Cell proteins or proteins were in and to for at the were and in phosphorylation in the presence or of 8-pCPT-cGMP at were with and by Western and COS-7 cells were with by the (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). of the the of calcium was performed using the IRAG is a of the in with InsP3R and InsP3-induced calcium release. It has been shown that IRAG and InsP3R are phosphorylated after of 8-pCPT-cGMP by cGKI. IRAG, and cGKI to a complex which has been purified from of bovine smooth muscle (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). the interaction and the interaction sites of IRAG with a yeast two-hybrid was performed using an IRAG lacking the and as bait and cGKI and cGKII as interaction was observed between IRAG and cGKIβ but interaction between IRAG and cGKIα or cGKII A and Further analysis showed that cGKIβ interacted with amino with IRAG. is of the and of cGKI in amino acids W. Hofmann F. PubMed Scopus Google Scholar). the region of only the containing the leucine zipper (1Francis S.H. Corbin J.D. Adv. Pharmacol. 1994; 26: 115-170Crossref PubMed Scopus (77) Google but not the T.M. Komalavilas P. Cornwell T.L. Hypertension. 1994; 23: 1141-1147Crossref PubMed Scopus (173) Google showed with IRAG we which of IRAG interacts with cGKIβ. study different IRAG were used as and full-length cGKIβ or cGKIβ as in the yeast two-hybrid system A The amino-terminal region containing the leucine zipper of cGKIβ interacted with the sequence between amino acids 152 and 184 of IRAG. sequence of IRAG contains amino acids of which amino acids are and The of cGKIβ has amino acids between the of the cGKIβ leucine zipper interaction between these sites might by electrostatic the domain present in the IRAG protein is not involved in the complex between IRAG and cGKIβ that site might the of IRAG with the The of the two-hybrid system were by the results obtained after in vivo expression of the various proteins in COS-7 The different cGKI cGKIα and were with full-length IRAG purified or proteins as in 9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar), we that IRAG, InsP3RI were expressed at Phosphorylation of IRAG and co-precipitation of the proteins was in IRAG was phosphorylated in the presence of cGKIβ and or not at all in the presence of co-precipitated only IRAG and cGKIβ but not IRAG and The of cGKIα to interact with IRAG was by to the IRAG Western showed that protein was expressed to a in the COS-7 these results we that phosphorylation of IRAG requires interaction of the amino-terminal region containing the leucine zipper of cGKIβ with IRAG. we investigated the of cGKIβ with the InsP3RI, the InsP3RI is phosphorylated by cGKI P. Lincoln T.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and is present in the The InsP3RI was with cGKIβ in the of IRAG in COS-7 did not InsP3RI with cGKIβ with a two-hybrid using cGKIβ as bait and different InsP3RI as preys. In interaction between these proteins not these results that cGKI and InsP3RI are not associated with However, IRAG was expressed with cGKIβ and InsP3RI all three proteins were co-precipitated or of the proteins were bound to the in the presence of cGKIβ. These results that IRAG the of these proteins in a potential cGKI phosphorylation sites been identified the IRAG protein (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). In the present the functional role of the phosphorylation of these by cGKI was In IRAG and expressed which is the IRAG 9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar) were phosphorylated by cGKIβ in the presence of 8-pCPT-cGMP in the to the expression the for cGKIβ and IRAG was in COS-7 phosphorylation reaction was specific for phosphorylation of was only observed cGKIβ was with IRAG The COS-7 expression system was used to study the phosphorylation of the phosphorylation the various phosphorylation sites and were to alanine by PCR. The and several were expressed in COS-7 cells with cGKIβ and phosphorylation of these proteins by cGKIβ was with type IRAG Mutation of Ser696 to alanine phosphorylation of IRAG to with the phosphorylation of the type protein In the phosphorylation of the was not As a of the phosphorylation that the phosphorylation reaction was between and The IRAG protein the of with the type or the IRAG protein the that Ser696 is a cGKIβ phosphorylation site in IRAG. The reduced phosphorylation of IRAG was by a expression of the IRAG protein by a reduced of cGKIβ for the proteins The of co-precipitation of cGKIβ and IRAG was using cGMP by phosphorylation and analysis. observed in the of IRAG type or proteins co-precipitated with indicating that the of the various did not the interaction of IRAG with cGKIβ. was the interaction site of IRAG did not of the However, phosphorylation was reduced in the protein These results that Ser696 is the cGKIβ phosphorylation Phosphorylation of IRAG by cGKIβ inhibits and InsP3-induced calcium release from intracellular stores in COS-7 cells (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). The phosphorylation for the inhibition were not that phosphorylation of the serines were involved in functional of cGKIβ. we or not inhibition of the calcium release was observed after of the IRAG constructs into COS-7 Western blot analysis that type IRAG and all proteins were expressed to the same in COS-7 A show and the of calcium of COS-7 cells with various IRAG and and cGKIβ. cGKIβ was by of 8-pCPT-cGMP the with In cells IRAG containing the and cGKIβ was to the calcium release. In cGKIβ to a the in cells IRAG containing the serines or type IRAG The the of to calcium in COS-7 cells with cGKIβ and IRAG or is in B. and showed as IRAG type decrease after activation of the reduced calcium release was in The same was obtained IRAG were The IRAG containing still a the in containing the and reduction of to calcium observed It from these results that phosphorylation of cGKIβ is essential for the of IRAG InsP3-induced calcium release. has been in various transduction pathways to of smooth inhibition of platelet and of of and the of (1Francis S.H. Corbin J.D. Adv. Pharmacol. 1994; 26: 115-170Crossref PubMed Scopus (77) Google Scholar, 2Lincoln T.M. Komalavilas P. Cornwell T.L. Hypertension. 1994; 23: 1141-1147Crossref PubMed Scopus (173) Google Scholar, 3Pfeifer A. Ruth P. Dostmann W. Sausbier M. Klatt P. Hofmann F. Rev. Physiol. Biochem. Pharmacol. 1999; 135: 105-149Crossref PubMed Google Scholar, 4Hofmann F. Ammendola A. Schlossmann J. J. Cell Sci. 2000; 113: 1671-1676Crossref PubMed Google Scholar, U. Rev. Physiol. Biochem. Pharmacol. 1999; 135: PubMed Google Scholar). of these are by different as shown by of the gene for cGKII and cGKI (8Pfeifer A. Klatt P. Massberg S. Ny L. Sausbier M. Hirneiß C. Wang G. Korth M. Aszódi A. Andersson E. Krombach F. Mayerhofer A. Ruth P. Fässler R. Hofmann F. EMBO J. 1998; 17: 3045-3051Crossref PubMed Scopus (459) Google Scholar, A. Aszódi A. U. Ruth P. Hofmann F. Fässler R. PubMed Scopus Google Scholar). proteins been identified that are phosphorylated by cGKI or and are involved in the regulation of the proposed These proteins cGMP S.H. Corbin J.D. Am. J. Physiol. 1998; Google Scholar), the of the channel A. Y. M. Hofmann F. Ruth P. P. L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), A Ruth P. Allescher H.D. J. Biol. Chem. 2000; Scholar), I. Somlyo A.V. Somlyo A.P. 2000; PubMed Scopus Google Scholar), E. K. M. D. J. U. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), A. Aszódi A. U. Ruth P. Hofmann F. Fässler R. PubMed Scopus Google Scholar, A. M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), the S. M. M. R. K. P. M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and IRAG (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). are proteins as the of I M. K. M. B. T. Y. S. T. Cell 1999; PubMed Scopus Google Scholar, Y. M. Lincoln T.M. Mendelsohn M.E. 1999; PubMed Scopus Google Scholar) and H. C. A. J. P. P. G. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that vivo for cGKI and smooth muscle tone in the of cytosolic IRAG has been identified as the substrate for cGKI that of smooth muscle by decreasing the calcium release from InsP3-sensitive stores (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (392) Google Scholar). IRAG was with InsP3R and cGKIβ. In we show that IRAG interacts only with the amino-terminal region containing the leucine zipper of cGKIβ and not with that of cGKIα or The IRAG sequence with the amino-terminal region containing the leucine zipper of cGKIβ not a phosphorylation site the that interaction between cGKIβ and IRAG is of the mechanism substrate interaction not activation of the kinase domain and in and of cGKIβ to IRAG the observed phosphorylation specificity for the and the of cGKIα to IRAG. The results of study to that cGMP in vivo by interaction of amino-terminal kinase with the substrate mechanism has been identified not only for the of cGKIβ and IRAG, but was with cGKII and of the of cGKII is for localization of the and the phosphorylation of A. M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it has been shown that cGKIα with amino-terminal leucine zipper to the of I Y. M. Lincoln T.M. Mendelsohn M.E. 1999; PubMed Scopus Google Scholar), the muscle K. H. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and the protein K. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In it that the kinase to a substrate sequence that is the phosphorylation The localization of the kinase to substrate a after in In to the kinase system it is that the of the cGMP not proteins as necessary for the kinase system K. J.D. 2000; PubMed Scopus Google Scholar). IRAG, and the InsP3RI cGKIβ and InsP3RI were not co-precipitated and did not interact in the two-hybrid screen. It is to that the of the complex expression of IRAG. In of is the that the InsP3RI phosphorylation was only cGKIβ was with the The of cGKI to the InsP3RI is in with in vivo cGKI phosphorylated in vivo the smooth muscle InsP3RI P. Lincoln T.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and calcium release in (17Tertyshnikova S. Yan X. Fein A. J. Physiol. ( Lond .). 1998; 512.1: 89-96Crossref Scopus (54) Google Scholar). is not in to the smooth muscle and of cGKIβ and IRAG which cGKIβ-dependent phosphorylation of IRAG and the for cGKIβ in smooth muscle of cGKIβ has been in the cytosolic A. Ruth P. Hofmann F. J. Biochem. PubMed Scopus Google Scholar). Phosphorylation of the InsP3RI by cGKIβ did not to the decrease in calcium release. the phosphorylation of Ser696 of IRAG. Mutation of Ser696 to alanine the of cGKIβ calcium release without of the The phosphorylation of the InsP3RI by kinase to the calcium release in vivo S. Fein A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) and an to smooth muscle are necessary to or not kinase smooth muscle via phosphorylation of the InsP3R and requires the presence of IRAG. It is possible that of the calcium release is specifically by were to the calcium release in type and cGKI smooth muscle cells (8Pfeifer A. Klatt P. Massberg S. Ny L. Sausbier M. Hirneiß C. Wang G. Korth M. Aszódi A. Andersson E. Krombach F. Mayerhofer A. Ruth P. Fässler R. Hofmann F. EMBO J. 1998; 17: 3045-3051Crossref PubMed Scopus (459) Google Scholar). with the functional obtained by intracellular calcium results that phosphorylation of Ser696 is for the of IRAG and cGKI InsP3-induced calcium release. The a functional the is involved in calcium the of the functional phosphorylation site of the cGKIβ substrate protein IRAG we the molecular mechanism by which cGKI inhibits calcium release in smooth to the role of InsP3R in for