Molecular Determinants of the Interaction between the Inositol 1,4,5-Trisphosphate Receptor-associated cGMP Kinase Substrate (IRAG) and cGMP Kinase Iβ
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m101530200
ISSN1083-351X
AutoresAldo Ammendola, Angela Geiselhöringer, Franz Hofmann, Jens Schlossmann,
Tópico(s)Phosphodiesterase function and regulation
ResumoCyclic 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 reporter 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 pEG202-LexA/bait plasmids, and their respective pJG4-5/prey plasmids were co-transformed by lithium acetate (21Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1734) Google Scholar) in the yeast strain EGY48. The expression of IRAG as protein was assayed by standard lysis of cells expressing appropriate constructs followed by SDS-PAGE and immunoblot analysis with antibodies directed against IRAG. Transformants were selected on synthetic dropout agar plates lacking tryptophan, leucine, and histidine. As defined protein interaction sites were investigated in this study, the performed screen was identical with the rescreen described by Ausubel et al.(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). Interaction results in activation of the reporter genes on selective media. Positive clones were identified by their blue color on 5-bromo-4-chloro-3-indoyl β-d-galactoside (X-Gal) plates and their ability to grow on media lacking leucine in the presence of galactose. Mutagenesis of IRAG phosphorylation sites from serine to alanine was performed by overlap extension PCR according to Ho et al. (22Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst .). 1989; 77: 51-59Crossref PubMed Scopus (7011) Google Scholar). For the PCR-based insertion of point mutations two complementary synthetic oligonucleotide primers carrying the mutation and a DNA fragment flanking primer pair are required. In a first PCR round, two corresponding DNA fragments were generated which were used together as template in a second PCR round with the flanking primer pair. The primer pairs used for the mutation of single phosphorylation sites (S1-S4) and the flanking primers (AJ1-AJ3) for the amplification of the complete IRAGa cDNA are listed in Table III of the supplement. Generation of multiple mutations was performed using single mutant plasmids as template DNA. AJ1/AJ2-generated IRAG amplicons were purified and ligated into EcoRI/BamHI-restricted pcDNA3.1 vector (Invitrogen) after digestion with the same enzymes and used for the in vitro phosphorylation assays. For construction of IRAG-GFP fusions used for Fura-2 AM calcium measurements AJ1/AJ3-amplified PCR products were cloned into theEcoRI/BamHI-restricted pEGFP-N3 vector (CLONTECH). All introduced mutations were confirmed by sequence analysis (ABI PrismTM Sequence analyzer, PerkinElmer Life Sciences). COS-7 cells grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) were transiently transfected with IRAG constructs cloned into pcDNA3.1 (for phosphorylation experiments) or pEGFP-N3 (for calcium measurements) by using the calcium phosphate method. After 48 h of incubation at 37 °C, cells were harvested for lysate preparation or directly used for calcium measurements, respectively. COS-7 cells transiently transfected with the different IRAG-pcDNA3.1 constructs, cGKIβ cloned in pMT3-vector or InsP3RI in pcDNA3 were washed twice with phosphate-buffered saline buffer and then harvested in phosphate-buffered saline using a cell scraper. Cells were centrifuged at 600 × g, washed with phosphate-buffered saline buffer, and then suspended in hypoosmotic lysis buffer (10 mm potassium phosphate, pH 7.4). Cells were then freeze-thawed, lysed using a syringe with a 4-gauge needle, and then frozen in aliquots at −70 °C. Lysates from COS-7 cells were assayed for IRAG, cGKIβ, and InsP3RI content using standard immunoblot analysis with specific antibodies. Cell lysates containing equal amounts of IRAG and cGKIβ were used for phosphorylation reactions. Cells (20–30 μg of protein) were incubated in 50 mm Mes, pH 6.9, 10 mm NaCl, 1 mm MgAc, 0.4 mm EGTA, 0.1% Lubrol-PX (Sigma), 0.1 mm [γ-32P]ATP (2,000 cpm/pmol, Amersham Pharmacia Biotech) in the presence or absence of 8-pCPT-cGMP (3 μm, Biolog) for 15 s up to 2 min at 30 °C. The reactions were stopped by addition of Laemmli buffer. Proteins were separated by SDS-PAGE and blotted to polyvinylidene difluoride membrane (Whatman). Incorporated radioactivity was visualized by autoradiography and phosphoimage analysis (BAS-1500, Fuji, Raytest). Quantification of phospholuminescence was performed using the AIDA 2.0 image analysis program. Cell lysate proteins or microsomal membrane proteins (200 μg) were solubilized in 4 mm MOPS, pH 7.4, 32 mm NaCl, 1.6% Lubrol-PX (20 min, 4 °C), centrifuged (11,000 × g, 10 min, 4 °C), and added to cGMP-agarose beads (Biolog). After incubation for 2 h at 4 °C the beads were washed and incubated in phosphorylation buffer in the presence or absence of 8-pCPT-cGMP (3 μm) (2 min at 30 °C). Proteins were eluted with Laemmli buffer and analyzed by SDS-PAGE, Western blot, and autoradiography. COS-7 cells were loaded with Fura-2 AM (5 μm, ICN) before measuring [Ca2+]i by the dual-wavelength microfluorescence technique (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). Analysis of the area under the curve of calcium transients was performed using the software program MicrocalTM Origin 6.0. IRAG is a new compound of the NO/cGMP signaling pathway which, in association with InsP3R and cGKIβ, negatively regulates InsP3-induced calcium release. It has been shown that IRAG and InsP3R are phosphorylated after addition of 8-pCPT-cGMP by cGKI. IRAG, InsP3R, and cGKI associate to a multimeric complex which has been purified from microsomal membranes of bovine tracheal 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). To analyze the interaction and identify the interaction sites of IRAG with cGK, a yeast two-hybrid screen was performed using an IRAG derivative (IRAG53–845) lacking the putative NH2- and COOH-terminal transmembrane domains as bait and cGKI variants and cGKII as preys (Fig.1). Strong interaction was observed between IRAG and cGKIβ but no detectable interaction between IRAG and cGKIα or cGKII (Fig. 1, A and B). Further analysis showed that cGKIβ interacted with its amino terminus with IRAG. This is of interest since the Iα and Iβ isozymes of cGKI differ in their first 100 amino acids (23Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (169) Google Scholar). Within the NH2-terminal region of cGKIβ, only the part containing the leucine zipper (cGKIβ1–53: cGKIβ-NT (1Francis S.H. Corbin J.D. Adv. Pharmacol. 1994; 26: 115-170Crossref PubMed Scopus (77) Google Scholar)) but not the linker (cGKIβ54–104: cGKIβ-NT (2Lincoln T.M. Komalavilas P. Cornwell T.L. Hypertension. 1994; 23: 1141-1147Crossref PubMed Scopus (173) Google Scholar)) showed association with IRAG (Fig. 1 B). Next, we analyzed which part of IRAG interacts with cGKIβ. For this study different IRAG variants were used as baits and full-length cGKIβ or cGKIβ variants as prey in the yeast two-hybrid system (Fig. 1, A andC). The amino-terminal region containing the leucine zipper of cGKIβ interacted with the peptide sequence between amino acids 152 and 184 of IRAG. This sequence of IRAG contains 33 amino acids of which 16 amino acids are charged (Asp, Glu, Lys, and Arg). The corresponding peptide of cGKIβ has 22 charged amino acids located between the leucine/isoleucine residues of the cGKIβ leucine zipper (Fig.1 D). Therefore, interaction between these two sites might be mediated by electrostatic interactions. Interestingly, the putative coiled-coil domain present in the IRAG protein is not involved in the complex formation between IRAG and cGKIβ suggesting that this site might mediate the assembly of IRAG with the InsP3RI. The interpretations of the two-hybrid system experiments were supported by the results obtained after in vivo expression of the various proteins in COS-7 cells. The two different cGKI isoforms, cGKIα and cGKIβ, were separately coexpressed with full-length IRAG and/or InsP3RI. Using partially purified or pure proteins as standard (for example, see Fig. 2a in Ref. 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 estimated that cGKIβ, IRAG, and/or InsP3RI were expressed at approximately equal amounts. Phosphorylation of IRAG and co-precipitation of the proteins was studied in cell lysates (Fig. 1 E). IRAG was phosphorylated heavily in the presence of cGKIβ and very weak or not at all in the presence of cGKIα. Furthermore, cGMP-agarose co-precipitated only IRAG and cGKIβ but not IRAG and cGKIα. The inability of cGKIα to interact with IRAG was caused by its inability to bind the IRAG protein, since regular Western blots showed that each protein was expressed to a similar level in the COS-7 cells. From these results we concluded that phosphorylation of IRAG requires interaction of the amino-terminal region containing the leucine zipper of cGKIβ with IRAG. Next we investigated the association of cGKIβ with the InsP3RI, since the InsP3RI is phosphorylated by cGKI (24Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 25Haug L.S. Jensen V. Hvalby O. Walaas S.I. Ostvold A.C. J. Biol. Chem. 1999; 274: 7467-7473Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and is present in the cGKI·IRAG macrocomplex. The InsP3RI was coexpressed with cGKIβ in the absence of IRAG in COS-7 cells. cGMP-agarose did not co-precipitate InsP3RI with cGKIβ (Fig. 1 E). This result agreed with a two-hybrid screen using cGKIβ as bait and different InsP3RI fragments as preys. In this screen no interaction between these proteins could be detected (data not shown). Therefore, these results indicate that cGKI and InsP3RI are not stably associated with each other. However, when IRAG was expressed together with cGKIβ and InsP3RI all three proteins were co-precipitated (Fig.1 E). 90% or more of the solubilized coexpressed proteins were bound to the cGMP-agarose in the presence of cGKIβ. These results suggest that IRAG could mediate the assembly of these proteins in a macrocomplex. Several potential cGKI phosphorylation sites have been identified previously within the IRAG protein (Fig.1 C) (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 study, the functional role of the phosphorylation of these serine residues by cGKI was analyzed. In control experiments, both native IRAG and expressed IRAGa which is ∼10 kDa larger than the native IRAG (see Ref. 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 cell lysates (Fig.2 A). Similar to the expression level, the stoichiometry for cGKIβ and IRAG was approximately equal in COS-7 cells. This phosphorylation reaction was specific for cGK, since cGMP-dependent phosphorylation of IRAGa was only observed when cGKIβ was coexpressed with IRAG (Fig. 2 A). The COS-7 expression system was used next to study the phosphorylation efficiency of the single phosphorylation sites. For this purpose, the various serine phosphorylation sites (at positions 118, 629, 683, and 696) were mutated to alanine (S118A = S1A; S629A = S2A; S683A = S3A; S696A = S4A) by PCR. The single mutants and several multiple mutants were transiently expressed in COS-7 cells together with cGKIβ and phosphorylation of these mutant proteins by cGKIβ was analyzed compared with wild type IRAG (Fig. 2 B). Mutation of Ser696 to alanine (S4A) diminished significantly cGMP-dependent phosphorylation of IRAG to 53.1 ± 5.9% (n = 5) when compared with the phosphorylation of the wild type protein (Fig. 2 B). In contrast, the phosphorylation efficiency of the triple mutant S123A was not affected being 126.1 ± 12.9% (n = 5). As expected, a time course of the phosphorylation indicated that the phosphorylation reaction was maximal between 1 and 2 min (Fig. 2 C). The IRAG S4A mutant protein incorporated about half the amount of phosphate compared with the wild type or the IRAG S123A mutant protein supporting the notion that Ser696 is a major cGKIβ phosphorylation site in IRAG. The reduced phosphorylation of IRAG S4A was neither caused by a decreased expression of the mutant IRAG protein nor by a reduced affinity of cGKIβ for the mutant proteins (Fig.3). The extent of co-precipitation of cGKIβ and IRAG was analyzed using cGMP affinity chromatography followed by phosphorylation and immunoblot analysis. No difference could be observed in the amount of IRAG wild type or mutant proteins co-precipitated with cGKIβ, indicating that the mutation of the various serine residues did not affect the interaction of IRAG with cGKIβ. This result was expected since the interaction site of IRAG did not include any of the mutated serines. However, phosphorylation was significantly reduced in the S4A mutant protein (Fig. 3). These results suggest that Ser696 is the predominant cGKIβ phosphorylation site. Phosphorylation of IRAG by cGKIβ inhibits bradykinin- 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 site(s) responsible for the inhibition were not identified. We anticipated that phosphorylation of the above mutated serines were involved in this functional effect of cGKIβ. Therefore, we tested whether or not cGMP-dependent inhibition of the calcium release was observed after transfection of the mutated IRAG constructs into COS-7 cells. Western blot analysis indicated that wild type IRAG and all mutated proteins were expressed to the same level in COS-7 cells. Fig. 4, A andB, show representative traces and the statistics, respectively, of bradykinin-induced calcium transients of COS-7 cells transfected with various IRAG mutants (S4A, S123A, and S1234A) and cGKIβ. cGKIβ was activated by addition of 8-pCPT-cGMP before the second stimulation with bradykinin. In cells expressing IRAG mutants containing the S4A mutation (S4A and S1234A), cGKIβ was unable to inhibit the calcium release. In contrast, active cGKIβ decreased to a similar extent the second Ca2+ peak in cells expressing IRAG containing the mutated serines S123A or wild type IRAG (Fig.4). The calculated area under the curve ratio of second to first bradykinin-induced calcium transient in COS-7 cells transfected with cGKIβ and IRAG single or multiple mutants is illustrated in Fig.4 B. While S1A, S2A, and S3A single mutants showed similar values as IRAG wild type (about 50–60% decrease after activation of cGKIβ), the reduced calcium release was abolished in mutant S4A. The same result was obtained when multiple-mutated IRAG variants were measured. The IRAG triple mutant containing simultaneously S123A mutations still mediated a decreased area under the curve ratio like wild type, whereas in multiple variants containing the S4A mutation (e.g. S34A and S1234A) no reduction of second to first calcium transient could be observed (Fig. 4 B). It can be concluded from these results that phosphorylation of Ser696by cGKIβ is essential for the inhibitory effect of IRAG on InsP3-induced calcium release. cGK has been implicated in various physiological signal transduction pathways leading to relaxation of smooth muscle, inhibition of platelet aggregation, cell motility and cell proliferation, secretion of intestinal fluid, bone growth, renin secretion, guidance of nerve fibers, and the development of synaptic plasticity (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, 26Eigenthaler M Lohmann S.M. Walter U. Pilz R.B. Rev. Physiol. Biochem. Pharmacol. 1999; 135: 173-209Crossref PubMed Google Scholar). Part of these functions are regulated by different cGK enzymes as shown by deletion 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, 27Pfeifer A. Aszódi A. Seidler U. Ruth P. Hofmann F. Fässler R. Science. 1996; 274: 2082-2086Crossref PubMed Scopus (344) Google Scholar). Only few proteins have been identified that are phosphorylated by cGKI or -II and are involved in the regulation of the proposed functions. These proteins include cGMP hydrolyzing phosphodiesterase 5 (28Wyatt T.A. Naftilan A.J. Francis S.H. Corbin J.D. Am. J. Physiol. 1998; 274: H448-455PubMed Google Scholar), the large subunit of the maxi-KCa channel (29Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 30Fukao M. Mason H.S. Britton F.C. Kenyon J.L. Horowitz B. Keef K.D. J. Biol. Chem. 1999; 274: 10927-10935Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), CRP2 (31Huber A Neuhuber W.L. Klugbauer N. Ruth P. Allescher H.D. J. Biol. Chem. 2000; 275: 5411-5504Google Scholar), telokin (32MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar), VASP (33Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), CFTR (27Pfeifer A. Aszódi A. Seidler U. Ruth P. Hofmann F. Fässler R. Science. 1996; 274: 2082-2086Crossref PubMed Scopus (344) Google Scholar, 34Vaandrager A.B. Smolenski A. Tilly B.C. Houtsmuller A.B. Ehlert E.M.E. Bot A.G.M. Edixhoven M. Boomaars W.E.M. Lohmann S.M. De Jonge H.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1466-1471Crossref PubMed Scopus (145) Google Scholar), the cerebellar G-substrate (35Endo S. Suzuki M. Sumi M. Nairn A.C. Morita R. Yamakawa K. Greengard P. Ito M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2467-2472Crossref PubMed Scopus (65) Google Scholar, 36Hall K.U. Collins S.P. Gamm D.M. Massa E. DePaoli-Roach A.A. Uhler M.D. J. Biol. Chem. 1999; 274: 3485-3495Abstract Full Text Full Text PDF PubMed Scopus (66) 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). There are additional proteins such as the myosin binding subunit of phosphatase I (37Nakamura 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, 38Surks 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 (448) Google Scholar) and RhoA (39Sauzeau 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 (523) Google Scholar) that may bein vivo substrates for cGKI and affect smooth muscle tone in the absence of elevated cytosolic Ca2+ concentrations. IRAG has been identified recently as the substrate for cGKI that mediates cGMP-dependent relaxation 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 co-purified together with InsP3R and cGKIβ. In this study, we show that IRAG interacts only with the amino-terminal region containing the leucine zipper of cGKIβ and not with that of cGKIα or cGKII. The IRAG sequence interacting with the amino-terminal region containing the leucine zipper of cGKIβ does not contain a phosphorylation site supporting the notion that interaction between cGKIβ and IRAG is independent of the mechanism allowing substrate binding. This interaction does not require activation of the kinase domain and should be stable in relaxed and contracted cells. Localization of cGKIβ to IRAG explains the observed phosphorylation specificity for the Iβ isozyme and the inability of cGKIα to modify IRAG. The results of this study contribute to earlier observations that cGMP kinases recognize their in vivo substrates by interaction of amino-terminal kinase sequences with the substrate protein. This mechanism has been identified not only for the co-localization of cGKIβ and IRAG, but was found also with cGKII and cGKIα. Myristoylation of the first glycine of cGKII is required for membrane localization of the enzyme and the phosphorylation of CFTR (34Vaandrager A.B. Smolenski A. Tilly B.C. Houtsmuller A.B. Ehlert E.M.E. Bot A.G.M. Edixhoven M. Boomaars W.E.M. Lohmann S.M. De Jonge H.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1466-1471Crossref PubMed Scopus (145) Google Scholar, 40Vaandrager A.B. Ehlert E.M.E. Jarchau T. Lohmann S.M. De Jonge H.R. J. Biol. Chem. 1996; 271: 7025-7029Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Furthermore, it has been shown that cGKIα binds with its unique amino-terminal leucine zipper to the myosin binding subunit of phosphatase I (38Surks 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 (448) Google Scholar), the skeletal muscle troponin T (41Yuasa K. Michibata H. Omori K. Yanaka N. J. Biol. Chem. 1999; 274: 37429-37434Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and the male germ cell-specific 42-kDa protein GKAP42 (42Yuasa K. Omori K. Yanaka N. J. Biol. Chem. 2000; 275: 4897-4905Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In each case it appears that the kinase binds to a substrate sequence that is outside the phosphorylation site. The permanent localization of the kinase to its substrate allows a rapid response after increases in cGMP. In comparison to the cAMP kinase system it is obvious that the subcellular organization of the cGMP kinases does not require extra anchoring proteins as necessary for the cAMP kinase signaling system (43Dodge K. Scott J.D. FEBS Lett. 2000; 476: 58-61Crossref PubMed Scopus (117) Google Scholar). IRAG, cGKIβ, and the InsP3RI could be co-purified together, whereas cGKIβ and InsP3RI were not co-precipitated and did not interact in the two-hybrid screen. It is therefore plausible to assume that the assembly of the triple complex required expression of IRAG. In support of this hypothesis is the finding that the InsP3RI phosphorylation was evident only when cGKIβ was coexpressed with the InsP3RI. The differential possibility of cGKI to phosphorylate the InsP3RI is in good agreement with previous in vivo findings. cGKI phosphorylated in vivo the smooth muscle InsP3RI (24Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and affected calcium release in megakaryocytes (17Tertyshnikova S. Yan X. Fein A. J. Physiol. ( Lond .). 1998; 512.1: 89-96Crossref Scopus (54) Google Scholar). This is not in contrast to the above results, since smooth muscle and platelets express high levels of cGKIβ and IRAG which apparently allows cGKIβ-dependent phosphorylation of IRAG and the InsP3RI. Possibly, additional substrates exist for cGKIβ in smooth muscle since ∼50% of cGKIβ immunreactivity has been found in the cytosolic fraction (44Keilbach A. Ruth P. Hofmann F. Eur. J. Biochem. 1992; 208: 467-473Crossref PubMed Scopus (101) Google Scholar). Phosphorylation of the InsP3RI by cGKIβ apparently did not contribute to the decrease in calcium release. This effect depended on the phosphorylation of Ser696 of IRAG. Mutation of Ser696 to alanine abolished complete the modulatory effect of cGKIβ on calcium release without disruption of the triple complex. The phosphorylation of the InsP3RI by cAMP kinase may be important to modulate the calcium release in vivo (45Tertyshnikova S. Fein A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1613-1617Crossref PubMed Scopus (91) Google Scholar) and may be an independent possibility to regulate smooth muscle contractility. Additional experiments are necessary to clarify whether or not cAMP kinase modulates smooth muscle contraction via phosphorylation of the InsP3R and requires the presence of IRAG. It is quite possible that modulation of the InsP3-dependent calcium release is specifically caused by cGKIβ, since cAMP analogs were unable to modulate the calcium release in murine aortic wild type and cGKI negative 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). Together with the functional data obtained by intracellular calcium measurements our results clearly demonstrate that phosphorylation of Ser696 is indispensable for the inhibitory effect of IRAG and cGKI on InsP3-induced calcium release. The data presented here add a further functional element how the NO/cGMP/cGKI signaling pathway is involved in calcium regulation. With the identification of the functional phosphorylation site of the cGKIβ substrate protein IRAG we threw more light on the molecular mechanism by which cGKI inhibits InsP3-dependent calcium release in smooth muscle. Subsequent work must aim to elucidate the role of InsP3R in this process more precisely. We thank Christine Wolf for excellent technical assistance.
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