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Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor Macromolecular Signaling Complex

Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 42, Issue of October 18, pp. 39397–39400, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor Macromolecular Signaling Complex* Received for publication, July 15, 2002, and in revised form, August 6, 2002 Published, JBC Papers in Press, August 7, 2002, DOI 10.1074/jbc.M207059200 Nikhil deSouza‡, Steven Reiken‡, Karol Ondrias§, Yi-ming Yang, Scot Matkovich, and Andrew R. Marks From the Center for Molecular Cardiology, Department of Medicine, and Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032 and the §Institute of Molecular Physiology, Slovak Academy of Science, Bratislava 83334, Slovak Republic kinase A (PKA) (1–7), non-receptor protein tyrosine kinases in The inositol 1,4,5-trisphosphate receptor (IP3R) is a 2 2 ubiquitously expressed intracellular calcium (Ca )re- the src family (8, 9), protein kinase C (10), Ca /calmodulin- lease channel on the endoplasmic reticulum. IP3Rs play dependent protein kinase II (10), and nitric oxide (NO)/cyclic- key roles in controlling Ca signals that activate nu- GMP-dependent protein kinase (11, 12) and are also autophos- merous cellular functions including T cell activation, phorylated (13). neurotransmitter release, oocyte fertilization and apo- PKA phosphorylates IP3R1 at serines 1589 and 1755. The ptosis. There are three forms of IP3R, all of which are neuronal type IP3R1, defined by inclusion of an alternatively ligand-gated channels activated by the second messen- spliced 40-amino acid exon located between the two PKA phos- ger inositol 1,4,5-trisphosphate. Channel function is phorylation sites, is preferentially phosphorylated at Ser-1755, modulated via cross-talk with other signaling pathways whereas the peripheral IP3R1 is phosphorylated more on Ser- including those mediated by kinases and phosphatases. 1589. IP3R2 and IP3R3 lack these consensus PKA phosphoryl- In particular IP3Rs are known to be regulated by cAMP- ation sites, although they do contain other putative PKA phos- dependent protein kinase (PKA) phosphorylation. In the phorylation sites. present study we show that PKA and the protein phos- Although initial studies have suggested that PKA phospho- phatases PP1 and PP2A are components of the IP3R1 rylation decreases IP3-induced Ca release (14, 15) these macromolecular signaling complex. PKA phosphoryla- studies may have been misinterpreted because of the confound- tion of IP3R1 increases channel activity in planar lipid ing opposing contribution of increased ER Ca pump (uptake) bilayers. These studies indicate that regulation of IP3R1 activity in response to PKA phosphorylation. PKA targeted to function via PKA phosphorylation involves components IP3R3 has been shown to decrease IP3-induced Ca release in of a macromolecular signaling complex. pancreatic acinar cells (6). In rat hepatocytes IP3-induced Ca oscillations were enhanced by cAMP (16). Additional studies have established that in fact PKA phosphorylation of IP3R1 Inositol 1,4,5-trisphosphate receptors (IP3R) are members increases IP3-induced Ca release by 2-fold (3). of the intracellular Ca release channel family that also in- Recently we showed that the cardiac and skeletal muscle cludes ryanodine receptors. These channels form the pathways ryanodine receptors (RyR1 and RyR2) are macromolecular sig- for Ca release that amplifies signals transmitted via plasma naling complexes that include PKA and phosphatases (PP1 for membrane receptors. The amplitude, frequency, and subcellu- RyR1; PP1 and PP2A for RyR2) (17, 18). We now show that lar localization of Ca signals play an important role in de- IP3R1 from rat cerebellum is also a macromolecular complex termining cellular responses. IP3Rs require the second mes- that includes PKA, PP2A, and PP1. senger inositol 1,4,5-trisphosphate (IP3) for activation but they are also regulated physiologically via cross-talk with other EXPERIMENTAL PROCEDURES signaling pathways. Among the signaling pathways that mod- Isolation of Rat Brain ER—Rat brain ER was prepared as described ulate IP3R function are those involving kinases and (37). Briefly, rat whole brain was minced and homogenized on ice, using phosphatases. a glass tissue homogenizer in buffer A (5 mM NaN ,1mM EDTA, 20 mM IP3Rs are phosphorylated by cyclic-AMP-dependent protein HEPES (pH 7.4), 5 g/ml aprotonin, 5 g/ml leupeptin, 1 mM benzami- dine, 10 g/ml pepstatin, and 1 mM pefabloc). Homogenate was centri- fuged for 15 min at 4,000  g. Supernatant was filtered through * This work was supported by Grants RO1 AI39794 and cheesecloth and centrifuged for 30 min at 90,000  g. Pellet was RO3 TW00949 (to A. R. M.) from the National Institutes of Health. The resuspended in buffer B (0.6 M KCl, 5 mM NaN ,20mM Na P O ,1mM 3 4 2 7 costs of publication of this article were defrayed in part by the payment EDTA, 10 mM HEPES (pH 7.2)), incubated on ice for 15 min, and of page charges. This article must therefore be hereby marked “adver- centrifuged at 4,000  g for 15 min. The resultant supernatant was tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate centrifuged at 90,000  g for 30 min. The pellet from the last spin was this fact. resuspended in buffer C (10% sucrose, 10 mM MOPS (pH 7.0)). ‡ These authors contributed equally. Immunoprecipitation and Phosphorylation of IP3R—Rat brain ER To whom correspondence should be addressed: Center for Molecular (200 g) was suspended in 0.5 ml of modified radioimmune precipita- Cardiology, Box 65, Columbia University College of Physicians & Sur- tion buffer (50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.5 mM NaF, 0.5 mM geons, Rm. 9-401, 630 West 168th Street, New York, NY 10032. Tel.: Na VO , 0.25% Triton X-100, and protease inhibitors) and incubated 3 4 212-305-0270; Fax: 212-305-3690; E-mail: [email protected]. with antibody overnight at 4 °C (IgG alone was used as negative control The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; for each antibody). Protein A-Sepharose beads were added, and the IP3, inositol 1,4,5-trisphosphate; PKA, protein kinase A; ER, endoplasmic mixture was incubated at 4 °C for 1 h, washed with 1 phosphorylation reticulum; RyR, ryanodine receptor; PP, phosphatase; MOPS, 4-morpho- linepropanesulfonic acid; PIPES, piperazine-N,N-bis(2-ethanesulfonic buffer (8 mM MgCl, 10 mM EGTA, 50 mM Tris, 50 mM PIPES (pH 6.8)), acid); LIZ, leucine/isoleucine zipper. and resuspended in 10 l of 1.5 phosphorylation buffer containing This paper is available on line at http://www.jbc.org 39397 This is an Open Access article under the CC BY license. 39398 IP3R Macromolecular Complex FIG.1. Phosphorylation of IP3R1 by cAMP-dependent PKA. Immunoprecipitated IP3R1 was phosphorylated with PKA (5 units) or cAMP (10 M). The PKA inhibitor PKI (500 nM) inhibited phospho- 5–24 rylation. Equivalent amounts of IP3R1 protein were used in each reac- tion as shown by the immunoblot. FIG.2. cAMP-dependent PKA is a component of the IP3R1 either vehicle alone, PKA catalytic subunit (Sigma), PKA plus PKA macromolecular complex. PKA was co-immunoprecipitated with inhibitor, PKI (Calbiochem), or cAMP as indicated. Phosphorylation of IP3R1 from 100 g of rat brain ER. In the first lane 10 g of rat brain the immunoprecipitated IP3R was initiated with MgATP (33 M) and ER was loaded as a positive control for the anti-PKA antibody (10% of [- P]ATP and terminated after 5 min (PKA catalytic subunit) or 15 the amount used for immunoprecipitation). PKA was immunoprecipi- tated with anti-PKA and anti-IP3R1 antibodies. IgG alone and pre- min (cAMP) at room temperature with the addition of 5 l of stop absorbed anti-IP3R1 antibodies (pre-absorbed with the antigenic pep- solution (4% SDS and 0.25 M dithiothreitol). Samples were heated to tide as described previously (35)) were used as negative controls. 95 °C and size-fractionated on 6% SDS-PAGE, and IP3R radioactivity was assessed with autoradiography. Microcystin-Sepharose (35 l, Upstate Biotechnology, Inc.) was used to isolate IP3R from 200 gofrat addition of phosphorylation buffer including cAMP without brain ER by incubating it at 4 °C for 4 h followed by washing with exogenous PKA also resulted in phosphorylation of IP3R1 that modified radioimmune precipitation buffer. Beads were resuspended in was inhibited by PKI, indicating that endogenous PKA was 6 SDS loading buffer and boiled. The supernatant was size-fraction- ated on SDS-PAGE. PP1 and PP2A bound to the microcystin-Sepharose associated with IP3R1 (Fig. 1). beads were competed off with free microcystin-LR (Calbiochem). The IP3R1 was co-immunoprecipitated from rat brain with the samples were washed three times with 1 ml of radioimmune precipita- catalytic subunit of PKA (Fig. 2). IP3R1. Also co-immunopre- tion buffer. Beads were resuspended in 6 loading buffer and boiled. cipitated with IP3R1 were two of the major protein phospha- The samples were size-fractionated on eithera6or12% SDS-PAGE. tases, protein phosphatase 2A (PP2A) and protein phosphatase Immunoblots were performed as described (9, 35) using anti-PP1 1 (PP1) (Fig. 3). These data are consistent with a high molecular (1:1000), anti-PKAc (1:1000 Transduction Labs Lexington, KY), or anti- IP3R1 (9, 35). After washing in Tris-buffered saline  0.1% Tween 20 weight complex comprising IP3R1, PKA, PP1, and PP2A. for 1 h, the blots were incubated with peroxidase-conjugated goat anti- The phosphatase inhibitor microcystin binds to PP1 and mouse or goat anti-rabbit (1:3000 Santa Cruz Biotechnology). After PP2A. IP3R1 was sedimented by binding to microcystin-Sepha- washing in Tris-buffered saline  0.1% Tween 20 for 1 h, the blots were rose beads; the specificity of this interaction was demonstrated developed using ECL (Amersham Biosciences). by competing off IP3R1 using free microcystin-LR (Fig. 4). Single Channel Recordings—Rat brain ER membranes (1 mg/ml) IP3R1 was also sedimented using cAMP-Sepharose beads that were fused with a black lipid membrane in a chamber similar to that described by Miller (36). Membranes were formed as described below bind to the regulatory subunit of PKA (RII) (Fig. 4). The spec- across a hole in a partition between two chambers. After membrane ificity of this reaction was demonstrated by competing off formation, vesicles were added to the one chamber (designated cis) IP3R1 using excess RII (Fig. 4). Taken together these data where they fused to the membrane. Dioleoyl-phosphatidylethanolamine show that PKA, RII, PP1, and PP2A comprise a macromolecu- and dioleoyl-phosphatidylserine (Avanti Polar Lipids) at a molar ratio 2 lar complex with IP3R1. of 3:1 were used as lipids. To measure the Ca current through the PKA phosphorylation of IP3R1 in planar lipid bilayers re- IP3R1 channel, cis and trans chambers were filled with the following solutions: cis solution, 250 mM Hepes, 113.8 mM Tris, 0.7 mM CaCl ,1 sulted in a significant increase in P from 0.0002 to 0.001 2 o mM EGTA, 0.5 mM MgCl ,50mM KCl, pH 7.4; trans solution, 53 mM 2 0.0002 (n  4/4, e.g. Fig. 5). In these experiments channel P is Ca(OH) , 0.5 mM MgCl , 250 mM Hepes, 50 mM KCl, pH 7.35. After 2 2 2 low after the addition of IP3 because the [Ca ] (cytoplasmic) cis formation of the bilayer membrane, native membrane vesicles were was 400 nM, a level at which IP3R1 channels have been shown added to the cis chamber while mixture was stirred. Fusion of the to have low activity (19). However, in one experiment a long membrane vesicles, proteoliposomes, or proteins with the bilayer was lasting open state was observed following PKA phosphorylation detected by step changes in membrane currents monitored under volt- age clamp conditions as described (9). After incorporation of the channel of an IP3R1 channel in the bilayer. These data show that PKA into the bilayer, the cis and/or trans chamber was perfused with the phosphorylation activates IP3R1 channels in planar lipid desired solutions. The cis chamber was connected to the head-stage bilayers. input of an Axon 200 amplifier (Axon Instruments) by an Ag/AgCl electrode and agar/KCl bridges. The trans chamber was held at ground DISCUSSION with a similar electrode. Single channel currents were continuously In the present study we show that neuronal IP3R1 is a monitored and recorded with a Digital Audio Tape (DAT) and/or a chart macromolecular complex that includes PKA, PP1, and PP2A. recorder. Recordings were filtered through a low-pass Bessel (Fre- quency Devices, Haverhill, MA) at 2 kHz and digitized at 0.5 kHz. There are two PKA phosphorylation sites on IP3R1, serines Single channel properties were analyzed using PClamp 6.01 software. 1589 and 1755 (1, 2, 5). Ser-1755 is the predominant PKA Data were collected for at least 3 min after the addition of channel phosphorylation site in neuronal IP3R1 (7). In non-neuronal modulators. In all experiments IP3 (5 M) was added after the mem- tissues, alternative splicing results in deletion of a 40-amino brane was established, and only channels that were activated by the acid exon (1) that affects PKA phosphorylation of the channel, addition of IP3 were studied. Control channels were activated by 5 M resulting in preferential phosphorylation of Ser-1589 in some IP3 in the presence of 100 M ATP in the cis solution. tissues (1). The data presented in this study suggest that the RESULTS phosphorylation of one or both of these sites is regulated by IP3R1 immunoprecipitated from rat brain was PKA-phos- components of the IP3R1 macromolecular complex in a manner phorylated in in vitro kinase reactions (Fig. 1). To confirm the similar to that previously shown for the closely related chan- identity of the PKA-phosphorylated high molecular weight pro- nels, type 1 and type 2 ryanodine receptors (RyR1 and RyR2), tein as IP3R1, the phosphorylated band was immunoblotted found in skeletal and cardiac muscle (17, 18), and for the with anti-IP3R1 antibody. The specificity of the phosphoryla- voltage-gated potassium channel KCNQ1 (20) and the voltage- tion was demonstrated using a PKA inhibitor (PKI, Fig. 1). The gated calcium channel (21). Therefore, IP3R1 can be added to IP3R Macromolecular Complex 39399 FIG.3. Two protein phosphatases, PP1 and PP2A, are compo- nents of the IP3R1 macromolecular complex. PP1 (upper panel) and PP2A (lower panel) were co-immunoprecipitated with IP3R1 from 100 g of rat brain ER. In the first lane in each blot 10 g of rat brain ER was loaded as a positive control for the anti-PP1 and anti-PP2A antibodies (10% of the amount used for immunoprecipitation). PP1 and PP2A were immunoprecipitated with anti-PP1 or anti-PP2A and anti- IP3R1 antibodies. IgG alone and pre-absorbed anti-IP3R1 antibodies (pre-absorbed with the antigenic peptide as described previously (35)) FIG.5. PKA phosphorylation activates IP3R1. A, single channel were used as negative controls. tracings showing activation of IP3R1 in planar lipid bilayer by IP3 (2 M) followed by further activation by the addition of PKA catalytic subunit (90 units) added to the cis (cytosolic) chamber. Channel open- ings are in the upward direction; the current amplitude for a fully open channel under these conditions was 2 pA. The bottom tracing shows the characteristic block of the IP3R1 channels by heparin (50 M). Recordings were at 0 mV potential across the lipid bilayer membrane; the lines at the left of each tracing indicate the closed state of the channels. Data shown are representative of three similar experiments. FIG.4. Kinase and phosphatase in the IP3R1 macromolecular (Tracings are from a single experiment with 400 nM [Ca] but are not cis complex. IP3R1 was pulled out from rat brain ER with either cAMP- continuous.) B, open probability (P ) of a single IP3R1 channel plotted Sepharose beads (binds PKA) or microcystin-Sepharose beads (binds as a function of time showing the effect of MgATP (0.5 mM), followed by PP1 and PP2A). Excess PKA regulatory subunit, which binds to the the addition of IP3, followed by the addition of PKA and finally heparin PKA catalytic subunit, blocked IP3R1 pull-down by cAMP beads, and (50 M). free microcystin-LR, which competes PP1 off the beads, blocked IP3R1 pull-down by microcystin beads. in the absence of many other cellular proteins, PKA phospho- rylation of the IP3R1 can activate the channel. The in vivo the list of ion channels that comprise macromolecular com- effect of PKA phosphorylation on net intracellular Ca release plexes designed to modulate channel activity in response to would of course reflect the activity of multiple molecules in- stimulation of adrenergic signaling pathways that reflect sym- volved in regulating Ca fluxes and is likely to be complex, as pathetic nervous system activity. shown in a variety of cellular systems (25, 26). In the present study we also show that IP3R1 channels in The finding that the IP3R1 macromolecular complex in- planar lipid bilayers can be activated by PKA phosphorylation cludes PKA, PP1, and PP2A points to the similarities between (Fig. 5). These data suggest that PKA phosphorylation of IP3R1 this complex and that of the closely related channels RyR1 and provides a mechanism for increasing channel activity, although RyR2 (18). We previously showed that the RyR1 and RyR2 extension of the in vitro studies presented in this paper to in macromolecular complexes involve targeting of kinases and vivo conditions may not apply to all situations. Indeed, previ- phosphatases to the channels via targeting proteins (18). In- ous studies have provided conflicting data showing that PKA terestingly, we showed that these targeting proteins contain phosphorylation of IP3R1 in vesicles either activates (3, 4, 22, leucine/isoleucine zippers (LIZs) that specifically bind to LIZs 23) or inhibits (14, 24) Ca flux. The conflicting conclusions on the RyRs (18). These LIZs are highly conserved between reached by these studies, all of which used Ca flux measure- RyR isoforms and through evolution (18). Moreover, the role of ments to infer the effects of PKA phosphorylation of IP3R1 on LIZs in the formation of ion channel macromolecular complexes channel function, underscore the importance of studies exam- is also present in voltage-gated channels including the potas- ining the effects of PKA phosphorylation directly on single sium channel KCNQ1 (20). channel function. Indeed, the effects of PKA phosphorylation We have identified three LIZ motifs that are highly con- on other proteins in the vesicles may explain the discrepancies served between IP3R isoforms. Based on the analogy to the between these studies. For example, PKA phosphorylation can RyR system, we would expect that these putative LIZs may activate Ca uptake pumps in vesicles, and this would de- form binding sites for targeting proteins that direct PKA, PP1, 2 2 crease Ca release. Thus, variations in the density of Ca and PP2A to IP3Rs. We are currently performing yeast two- pumps in the vesicles could explain why one group finds PKA- hybrid assays to identify potential targeting proteins. Only one induced increased Ca release and another group finds a of the three putative LIZs shares homology with one of the RyR decrease. A direct examination of single channel currents in LIZs; this sequence is homologous to the muscle A-kinase- planar lipid bilayers in the present study suggests that, at least anchoring protein (mAKAP) binding site on RyR2. 39400 IP3R Macromolecular Complex 13. Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. The IP3R1 macromolecular complex contains additional pro- (1992) J. Biol. Chem. 267, 7036 –7041 teins beyond those described in the present study, including: 14. Supattapone, S., Danoff, S. K., Theibert, A., Joseph, S. K., Steiner, J., and Snyder, S. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8747– 8750 calmodulin (27), Homer (28), caldendrin (29), and FKBP12 and 15. Tertyshnikova, S., and Fein, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, calcineurin (24, 30, 31) (although there are conflicting reports 1613–1617 (32–34)). Moreover, it is highly likely that additional compo- 16. Chatton, J. Y., Cao, Y., Liu, H., and Stucki, J. W. (1998) Biochem. J. 330, 1411–1416 nents of the IP3R1 macromolecular complex will be added to 17. Marx, S. O., Reiken, S., Hisamatsu, Y., Jayaraman, T., Burkhoff, D., Rosemblit, this growing list. N., and Marks, A. R. (2000) Cell 101, 365–376 18. Marx, S. O., Reiken, S., Hisamatsu, Y., Gaburjakova, M., Gaburjakova, J., In the present study we have shown, using two independent Yang, Y. M., Rosemblit, N., and Marks, A. R. (2001) J. Cell Biol. 153, methods (co-immunoprecipitation and cAMP-Sepharose and 699 –708 microcystin-Sepharose pull-downs), that IP3R1 is a macromo- 19. Bezprozvanny, I., Watras, J., and Ehrlich, B. (1991) Nature 351, 751–754 20. Marx, S. O., Kurokawa, J., Reiken, S., Motoike, H., D’Armiento, J., Marks, lecular complex that includes PKA, PP1, and PP2A. We have A. R., and Kass, R. S. (2002) Science 295, 496 – 499 also shown that PKA phosphorylation activates IP3R1 at the 21. Hulme, J. T., Ahn, M., Hauschka, S. D., Scheuer, T., and Catterall, W. A. (2002) J. Biol. Chem. 277, 4079 – 4087 single channel level. Defining the components of the IP3R1 22. Volpe, P., and Alderson-Lang, B. H. (1990) Am. J. Physiol. 258, C1086 –C1091 macromolecular signaling complex provides important infor- 23. Hajnoczky, G., Gao, E., Nomura, T., Hoek, J. B., and Thomas, A. P. (1993) mation that can be used to characterize further the physiolog- Biochem. J. 293, 413– 422 24. Cameron, A. M., Steiner, J. P., Roskams, A. J., Ali, S. M., Ronnett, G. V., and ical role of modulation of IP3R1 by PKA phosphorylation. Snyder, S. H. (1995) Cell 83, 463– 472 25. Straub, S. V., Giovannucci, D. R., Bruce, J. 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Komalavilas, P., and Lincoln, T. M. (1996) J. Biol. Chem. 271, 21933–21938 111, 847– 856 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor Macromolecular Signaling Complex

deSouza, Nikhil; Reiken, Steven; Ondrias, Karol; Yang, Yi-ming; Matkovich, Scot; Marks, Andrew R.
Journal of Biological Chemistry – Oct 1, 2002
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0021-9258
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10.1074/jbc.m207059200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 42, Issue of October 18, pp. 39397–39400, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein Kinase A and Two Phosphatases Are Components of the Inositol 1,4,5-Trisphosphate Receptor Macromolecular Signaling Complex* Received for publication, July 15, 2002, and in revised form, August 6, 2002 Published, JBC Papers in Press, August 7, 2002, DOI 10.1074/jbc.M207059200 Nikhil deSouza‡, Steven Reiken‡, Karol Ondrias§, Yi-ming Yang, Scot Matkovich, and Andrew R. Marks From the Center for Molecular Cardiology, Department of Medicine, and Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032 and the §Institute of Molecular Physiology, Slovak Academy of Science, Bratislava 83334, Slovak Republic kinase A (PKA) (1–7), non-receptor protein tyrosine kinases in The inositol 1,4,5-trisphosphate receptor (IP3R) is a 2 2 ubiquitously expressed intracellular calcium (Ca )re- the src family (8, 9), protein kinase C (10), Ca /calmodulin- lease channel on the endoplasmic reticulum. IP3Rs play dependent protein kinase II (10), and nitric oxide (NO)/cyclic- key roles in controlling Ca signals that activate nu- GMP-dependent protein kinase (11, 12) and are also autophos- merous cellular functions including T cell activation, phorylated (13). neurotransmitter release, oocyte fertilization and apo- PKA phosphorylates IP3R1 at serines 1589 and 1755. The ptosis. There are three forms of IP3R, all of which are neuronal type IP3R1, defined by inclusion of an alternatively ligand-gated channels activated by the second messen- spliced 40-amino acid exon located between the two PKA phos- ger inositol 1,4,5-trisphosphate. Channel function is phorylation sites, is preferentially phosphorylated at Ser-1755, modulated via cross-talk with other signaling pathways whereas the peripheral IP3R1 is phosphorylated more on Ser- including those mediated by kinases and phosphatases. 1589. IP3R2 and IP3R3 lack these consensus PKA phosphoryl- In particular IP3Rs are known to be regulated by cAMP- ation sites, although they do contain other putative PKA phos- dependent protein kinase (PKA) phosphorylation. In the phorylation sites. present study we show that PKA and the protein phos- Although initial studies have suggested that PKA phospho- phatases PP1 and PP2A are components of the IP3R1 rylation decreases IP3-induced Ca release (14, 15) these macromolecular signaling complex. PKA phosphoryla- studies may have been misinterpreted because of the confound- tion of IP3R1 increases channel activity in planar lipid ing opposing contribution of increased ER Ca pump (uptake) bilayers. These studies indicate that regulation of IP3R1 activity in response to PKA phosphorylation. PKA targeted to function via PKA phosphorylation involves components IP3R3 has been shown to decrease IP3-induced Ca release in of a macromolecular signaling complex. pancreatic acinar cells (6). In rat hepatocytes IP3-induced Ca oscillations were enhanced by cAMP (16). Additional studies have established that in fact PKA phosphorylation of IP3R1 Inositol 1,4,5-trisphosphate receptors (IP3R) are members increases IP3-induced Ca release by 2-fold (3). of the intracellular Ca release channel family that also in- Recently we showed that the cardiac and skeletal muscle cludes ryanodine receptors. These channels form the pathways ryanodine receptors (RyR1 and RyR2) are macromolecular sig- for Ca release that amplifies signals transmitted via plasma naling complexes that include PKA and phosphatases (PP1 for membrane receptors. The amplitude, frequency, and subcellu- RyR1; PP1 and PP2A for RyR2) (17, 18). We now show that lar localization of Ca signals play an important role in de- IP3R1 from rat cerebellum is also a macromolecular complex termining cellular responses. IP3Rs require the second mes- that includes PKA, PP2A, and PP1. senger inositol 1,4,5-trisphosphate (IP3) for activation but they are also regulated physiologically via cross-talk with other EXPERIMENTAL PROCEDURES signaling pathways. Among the signaling pathways that mod- Isolation of Rat Brain ER—Rat brain ER was prepared as described ulate IP3R function are those involving kinases and (37). Briefly, rat whole brain was minced and homogenized on ice, using phosphatases. a glass tissue homogenizer in buffer A (5 mM NaN ,1mM EDTA, 20 mM IP3Rs are phosphorylated by cyclic-AMP-dependent protein HEPES (pH 7.4), 5 g/ml aprotonin, 5 g/ml leupeptin, 1 mM benzami- dine, 10 g/ml pepstatin, and 1 mM pefabloc). Homogenate was centri- fuged for 15 min at 4,000  g. Supernatant was filtered through * This work was supported by Grants RO1 AI39794 and cheesecloth and centrifuged for 30 min at 90,000  g. Pellet was RO3 TW00949 (to A. R. M.) from the National Institutes of Health. The resuspended in buffer B (0.6 M KCl, 5 mM NaN ,20mM Na P O ,1mM 3 4 2 7 costs of publication of this article were defrayed in part by the payment EDTA, 10 mM HEPES (pH 7.2)), incubated on ice for 15 min, and of page charges. This article must therefore be hereby marked “adver- centrifuged at 4,000  g for 15 min. The resultant supernatant was tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate centrifuged at 90,000  g for 30 min. The pellet from the last spin was this fact. resuspended in buffer C (10% sucrose, 10 mM MOPS (pH 7.0)). ‡ These authors contributed equally. Immunoprecipitation and Phosphorylation of IP3R—Rat brain ER To whom correspondence should be addressed: Center for Molecular (200 g) was suspended in 0.5 ml of modified radioimmune precipita- Cardiology, Box 65, Columbia University College of Physicians & Sur- tion buffer (50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.5 mM NaF, 0.5 mM geons, Rm. 9-401, 630 West 168th Street, New York, NY 10032. Tel.: Na VO , 0.25% Triton X-100, and protease inhibitors) and incubated 3 4 212-305-0270; Fax: 212-305-3690; E-mail: [email protected]. with antibody overnight at 4 °C (IgG alone was used as negative control The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; for each antibody). Protein A-Sepharose beads were added, and the IP3, inositol 1,4,5-trisphosphate; PKA, protein kinase A; ER, endoplasmic mixture was incubated at 4 °C for 1 h, washed with 1 phosphorylation reticulum; RyR, ryanodine receptor; PP, phosphatase; MOPS, 4-morpho- linepropanesulfonic acid; PIPES, piperazine-N,N-bis(2-ethanesulfonic buffer (8 mM MgCl, 10 mM EGTA, 50 mM Tris, 50 mM PIPES (pH 6.8)), acid); LIZ, leucine/isoleucine zipper. and resuspended in 10 l of 1.5 phosphorylation buffer containing This paper is available on line at http://www.jbc.org 39397 This is an Open Access article under the CC BY license. 39398 IP3R Macromolecular Complex FIG.1. Phosphorylation of IP3R1 by cAMP-dependent PKA. Immunoprecipitated IP3R1 was phosphorylated with PKA (5 units) or cAMP (10 M). The PKA inhibitor PKI (500 nM) inhibited phospho- 5–24 rylation. Equivalent amounts of IP3R1 protein were used in each reac- tion as shown by the immunoblot. FIG.2. cAMP-dependent PKA is a component of the IP3R1 either vehicle alone, PKA catalytic subunit (Sigma), PKA plus PKA macromolecular complex. PKA was co-immunoprecipitated with inhibitor, PKI (Calbiochem), or cAMP as indicated. Phosphorylation of IP3R1 from 100 g of rat brain ER. In the first lane 10 g of rat brain the immunoprecipitated IP3R was initiated with MgATP (33 M) and ER was loaded as a positive control for the anti-PKA antibody (10% of [- P]ATP and terminated after 5 min (PKA catalytic subunit) or 15 the amount used for immunoprecipitation). PKA was immunoprecipi- tated with anti-PKA and anti-IP3R1 antibodies. IgG alone and pre- min (cAMP) at room temperature with the addition of 5 l of stop absorbed anti-IP3R1 antibodies (pre-absorbed with the antigenic pep- solution (4% SDS and 0.25 M dithiothreitol). Samples were heated to tide as described previously (35)) were used as negative controls. 95 °C and size-fractionated on 6% SDS-PAGE, and IP3R radioactivity was assessed with autoradiography. Microcystin-Sepharose (35 l, Upstate Biotechnology, Inc.) was used to isolate IP3R from 200 gofrat addition of phosphorylation buffer including cAMP without brain ER by incubating it at 4 °C for 4 h followed by washing with exogenous PKA also resulted in phosphorylation of IP3R1 that modified radioimmune precipitation buffer. Beads were resuspended in was inhibited by PKI, indicating that endogenous PKA was 6 SDS loading buffer and boiled. The supernatant was size-fraction- ated on SDS-PAGE. PP1 and PP2A bound to the microcystin-Sepharose associated with IP3R1 (Fig. 1). beads were competed off with free microcystin-LR (Calbiochem). The IP3R1 was co-immunoprecipitated from rat brain with the samples were washed three times with 1 ml of radioimmune precipita- catalytic subunit of PKA (Fig. 2). IP3R1. Also co-immunopre- tion buffer. Beads were resuspended in 6 loading buffer and boiled. cipitated with IP3R1 were two of the major protein phospha- The samples were size-fractionated on eithera6or12% SDS-PAGE. tases, protein phosphatase 2A (PP2A) and protein phosphatase Immunoblots were performed as described (9, 35) using anti-PP1 1 (PP1) (Fig. 3). These data are consistent with a high molecular (1:1000), anti-PKAc (1:1000 Transduction Labs Lexington, KY), or anti- IP3R1 (9, 35). After washing in Tris-buffered saline  0.1% Tween 20 weight complex comprising IP3R1, PKA, PP1, and PP2A. for 1 h, the blots were incubated with peroxidase-conjugated goat anti- The phosphatase inhibitor microcystin binds to PP1 and mouse or goat anti-rabbit (1:3000 Santa Cruz Biotechnology). After PP2A. IP3R1 was sedimented by binding to microcystin-Sepha- washing in Tris-buffered saline  0.1% Tween 20 for 1 h, the blots were rose beads; the specificity of this interaction was demonstrated developed using ECL (Amersham Biosciences). by competing off IP3R1 using free microcystin-LR (Fig. 4). Single Channel Recordings—Rat brain ER membranes (1 mg/ml) IP3R1 was also sedimented using cAMP-Sepharose beads that were fused with a black lipid membrane in a chamber similar to that described by Miller (36). Membranes were formed as described below bind to the regulatory subunit of PKA (RII) (Fig. 4). The spec- across a hole in a partition between two chambers. After membrane ificity of this reaction was demonstrated by competing off formation, vesicles were added to the one chamber (designated cis) IP3R1 using excess RII (Fig. 4). Taken together these data where they fused to the membrane. Dioleoyl-phosphatidylethanolamine show that PKA, RII, PP1, and PP2A comprise a macromolecu- and dioleoyl-phosphatidylserine (Avanti Polar Lipids) at a molar ratio 2 lar complex with IP3R1. of 3:1 were used as lipids. To measure the Ca current through the PKA phosphorylation of IP3R1 in planar lipid bilayers re- IP3R1 channel, cis and trans chambers were filled with the following solutions: cis solution, 250 mM Hepes, 113.8 mM Tris, 0.7 mM CaCl ,1 sulted in a significant increase in P from 0.0002 to 0.001 2 o mM EGTA, 0.5 mM MgCl ,50mM KCl, pH 7.4; trans solution, 53 mM 2 0.0002 (n  4/4, e.g. Fig. 5). In these experiments channel P is Ca(OH) , 0.5 mM MgCl , 250 mM Hepes, 50 mM KCl, pH 7.35. After 2 2 2 low after the addition of IP3 because the [Ca ] (cytoplasmic) cis formation of the bilayer membrane, native membrane vesicles were was 400 nM, a level at which IP3R1 channels have been shown added to the cis chamber while mixture was stirred. Fusion of the to have low activity (19). However, in one experiment a long membrane vesicles, proteoliposomes, or proteins with the bilayer was lasting open state was observed following PKA phosphorylation detected by step changes in membrane currents monitored under volt- age clamp conditions as described (9). After incorporation of the channel of an IP3R1 channel in the bilayer. These data show that PKA into the bilayer, the cis and/or trans chamber was perfused with the phosphorylation activates IP3R1 channels in planar lipid desired solutions. The cis chamber was connected to the head-stage bilayers. input of an Axon 200 amplifier (Axon Instruments) by an Ag/AgCl electrode and agar/KCl bridges. The trans chamber was held at ground DISCUSSION with a similar electrode. Single channel currents were continuously In the present study we show that neuronal IP3R1 is a monitored and recorded with a Digital Audio Tape (DAT) and/or a chart macromolecular complex that includes PKA, PP1, and PP2A. recorder. Recordings were filtered through a low-pass Bessel (Fre- quency Devices, Haverhill, MA) at 2 kHz and digitized at 0.5 kHz. There are two PKA phosphorylation sites on IP3R1, serines Single channel properties were analyzed using PClamp 6.01 software. 1589 and 1755 (1, 2, 5). Ser-1755 is the predominant PKA Data were collected for at least 3 min after the addition of channel phosphorylation site in neuronal IP3R1 (7). In non-neuronal modulators. In all experiments IP3 (5 M) was added after the mem- tissues, alternative splicing results in deletion of a 40-amino brane was established, and only channels that were activated by the acid exon (1) that affects PKA phosphorylation of the channel, addition of IP3 were studied. Control channels were activated by 5 M resulting in preferential phosphorylation of Ser-1589 in some IP3 in the presence of 100 M ATP in the cis solution. tissues (1). The data presented in this study suggest that the RESULTS phosphorylation of one or both of these sites is regulated by IP3R1 immunoprecipitated from rat brain was PKA-phos- components of the IP3R1 macromolecular complex in a manner phorylated in in vitro kinase reactions (Fig. 1). To confirm the similar to that previously shown for the closely related chan- identity of the PKA-phosphorylated high molecular weight pro- nels, type 1 and type 2 ryanodine receptors (RyR1 and RyR2), tein as IP3R1, the phosphorylated band was immunoblotted found in skeletal and cardiac muscle (17, 18), and for the with anti-IP3R1 antibody. The specificity of the phosphoryla- voltage-gated potassium channel KCNQ1 (20) and the voltage- tion was demonstrated using a PKA inhibitor (PKI, Fig. 1). The gated calcium channel (21). Therefore, IP3R1 can be added to IP3R Macromolecular Complex 39399 FIG.3. Two protein phosphatases, PP1 and PP2A, are compo- nents of the IP3R1 macromolecular complex. PP1 (upper panel) and PP2A (lower panel) were co-immunoprecipitated with IP3R1 from 100 g of rat brain ER. In the first lane in each blot 10 g of rat brain ER was loaded as a positive control for the anti-PP1 and anti-PP2A antibodies (10% of the amount used for immunoprecipitation). PP1 and PP2A were immunoprecipitated with anti-PP1 or anti-PP2A and anti- IP3R1 antibodies. IgG alone and pre-absorbed anti-IP3R1 antibodies (pre-absorbed with the antigenic peptide as described previously (35)) FIG.5. PKA phosphorylation activates IP3R1. A, single channel were used as negative controls. tracings showing activation of IP3R1 in planar lipid bilayer by IP3 (2 M) followed by further activation by the addition of PKA catalytic subunit (90 units) added to the cis (cytosolic) chamber. Channel open- ings are in the upward direction; the current amplitude for a fully open channel under these conditions was 2 pA. The bottom tracing shows the characteristic block of the IP3R1 channels by heparin (50 M). Recordings were at 0 mV potential across the lipid bilayer membrane; the lines at the left of each tracing indicate the closed state of the channels. Data shown are representative of three similar experiments. FIG.4. Kinase and phosphatase in the IP3R1 macromolecular (Tracings are from a single experiment with 400 nM [Ca] but are not cis complex. IP3R1 was pulled out from rat brain ER with either cAMP- continuous.) B, open probability (P ) of a single IP3R1 channel plotted Sepharose beads (binds PKA) or microcystin-Sepharose beads (binds as a function of time showing the effect of MgATP (0.5 mM), followed by PP1 and PP2A). Excess PKA regulatory subunit, which binds to the the addition of IP3, followed by the addition of PKA and finally heparin PKA catalytic subunit, blocked IP3R1 pull-down by cAMP beads, and (50 M). free microcystin-LR, which competes PP1 off the beads, blocked IP3R1 pull-down by microcystin beads. in the absence of many other cellular proteins, PKA phospho- rylation of the IP3R1 can activate the channel. The in vivo the list of ion channels that comprise macromolecular com- effect of PKA phosphorylation on net intracellular Ca release plexes designed to modulate channel activity in response to would of course reflect the activity of multiple molecules in- stimulation of adrenergic signaling pathways that reflect sym- volved in regulating Ca fluxes and is likely to be complex, as pathetic nervous system activity. shown in a variety of cellular systems (25, 26). In the present study we also show that IP3R1 channels in The finding that the IP3R1 macromolecular complex in- planar lipid bilayers can be activated by PKA phosphorylation cludes PKA, PP1, and PP2A points to the similarities between (Fig. 5). These data suggest that PKA phosphorylation of IP3R1 this complex and that of the closely related channels RyR1 and provides a mechanism for increasing channel activity, although RyR2 (18). We previously showed that the RyR1 and RyR2 extension of the in vitro studies presented in this paper to in macromolecular complexes involve targeting of kinases and vivo conditions may not apply to all situations. Indeed, previ- phosphatases to the channels via targeting proteins (18). In- ous studies have provided conflicting data showing that PKA terestingly, we showed that these targeting proteins contain phosphorylation of IP3R1 in vesicles either activates (3, 4, 22, leucine/isoleucine zippers (LIZs) that specifically bind to LIZs 23) or inhibits (14, 24) Ca flux. The conflicting conclusions on the RyRs (18). These LIZs are highly conserved between reached by these studies, all of which used Ca flux measure- RyR isoforms and through evolution (18). Moreover, the role of ments to infer the effects of PKA phosphorylation of IP3R1 on LIZs in the formation of ion channel macromolecular complexes channel function, underscore the importance of studies exam- is also present in voltage-gated channels including the potas- ining the effects of PKA phosphorylation directly on single sium channel KCNQ1 (20). channel function. Indeed, the effects of PKA phosphorylation We have identified three LIZ motifs that are highly con- on other proteins in the vesicles may explain the discrepancies served between IP3R isoforms. Based on the analogy to the between these studies. For example, PKA phosphorylation can RyR system, we would expect that these putative LIZs may activate Ca uptake pumps in vesicles, and this would de- form binding sites for targeting proteins that direct PKA, PP1, 2 2 crease Ca release. Thus, variations in the density of Ca and PP2A to IP3Rs. We are currently performing yeast two- pumps in the vesicles could explain why one group finds PKA- hybrid assays to identify potential targeting proteins. Only one induced increased Ca release and another group finds a of the three putative LIZs shares homology with one of the RyR decrease. A direct examination of single channel currents in LIZs; this sequence is homologous to the muscle A-kinase- planar lipid bilayers in the present study suggests that, at least anchoring protein (mAKAP) binding site on RyR2. 39400 IP3R Macromolecular Complex 13. Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. The IP3R1 macromolecular complex contains additional pro- (1992) J. Biol. Chem. 267, 7036 –7041 teins beyond those described in the present study, including: 14. Supattapone, S., Danoff, S. K., Theibert, A., Joseph, S. K., Steiner, J., and Snyder, S. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8747– 8750 calmodulin (27), Homer (28), caldendrin (29), and FKBP12 and 15. Tertyshnikova, S., and Fein, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, calcineurin (24, 30, 31) (although there are conflicting reports 1613–1617 (32–34)). Moreover, it is highly likely that additional compo- 16. Chatton, J. Y., Cao, Y., Liu, H., and Stucki, J. W. (1998) Biochem. J. 330, 1411–1416 nents of the IP3R1 macromolecular complex will be added to 17. Marx, S. O., Reiken, S., Hisamatsu, Y., Jayaraman, T., Burkhoff, D., Rosemblit, this growing list. N., and Marks, A. R. (2000) Cell 101, 365–376 18. Marx, S. O., Reiken, S., Hisamatsu, Y., Gaburjakova, M., Gaburjakova, J., In the present study we have shown, using two independent Yang, Y. M., Rosemblit, N., and Marks, A. R. (2001) J. Cell Biol. 153, methods (co-immunoprecipitation and cAMP-Sepharose and 699 –708 microcystin-Sepharose pull-downs), that IP3R1 is a macromo- 19. Bezprozvanny, I., Watras, J., and Ehrlich, B. (1991) Nature 351, 751–754 20. Marx, S. O., Kurokawa, J., Reiken, S., Motoike, H., D’Armiento, J., Marks, lecular complex that includes PKA, PP1, and PP2A. We have A. R., and Kass, R. S. (2002) Science 295, 496 – 499 also shown that PKA phosphorylation activates IP3R1 at the 21. Hulme, J. T., Ahn, M., Hauschka, S. D., Scheuer, T., and Catterall, W. A. (2002) J. Biol. Chem. 277, 4079 – 4087 single channel level. Defining the components of the IP3R1 22. Volpe, P., and Alderson-Lang, B. H. (1990) Am. J. Physiol. 258, C1086 –C1091 macromolecular signaling complex provides important infor- 23. Hajnoczky, G., Gao, E., Nomura, T., Hoek, J. B., and Thomas, A. P. (1993) mation that can be used to characterize further the physiolog- Biochem. J. 293, 413– 422 24. Cameron, A. M., Steiner, J. P., Roskams, A. J., Ali, S. M., Ronnett, G. V., and ical role of modulation of IP3R1 by PKA phosphorylation. Snyder, S. H. (1995) Cell 83, 463– 472 25. Straub, S. V., Giovannucci, D. R., Bruce, J. 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