Aug 26, 1991 - receptor is regulated by ATP, calcium, and phos- phorylation by protein ... inositol 1,4,5-trisphosphate receptor; PKA, protein kinase A; PKC,.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 267, No. 10,Issue of April 5, pp. 7036-7041, 1992
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Autophosphorylation of Inositol 1,4,5-Trisphosphate Receptors* (Received for publication, August 26, 1991)
Christopher D. Ferris$, AndrewM. Cameron$, DavidS . BredtQ, Richard L. Huganir$V, and Solomon H. Snyder$Qll** From the $Departments of Neuroscience, $Pharmacologyand Molecular Sciences, 1lPsychiatt-y and Behavioral Sciences, TThe Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Inositol1,4,S-trisphosphate(IP,)releasesinternal mediate IP,-dependent activation of calcium ion channel acstores of calcium by binding to a specific membrane tivity (13). receptor which includes both the IP, recognition site Multiple forms of IP3R are generated through alternative as well as theassociatedcalciumchannel.The IP, splicing. A 15-amino acid insert (11)lies near the N terminus receptor is regulatedbyATP,calcium,andphoswhere IP3binding may take place (11,14,15). A second splice phorylation by protein kinase A, protein kinaseC, and calcium/calmodulin-dependentprotein kinase 11. Its site, in the middle third of the sequence (16, 17), is 40 amino cDNAsequencepredicts at least two consensus se- acids long and appears to regulate the specificity of protein kinase A (PKA) phosphorylation of the IP3R (16). Splicing quences where nucleotides might bind, and direct binding of ATP to the IP, receptor has been demonstrated.in this regionmayinvolve four different mRNAs through In the present study, we demonstrate autophosphory- variations in this 40-amino acid sequence (17). Further hetmay result from the expression of multiple genes lation of the purified and reconstituted IP, receptor erogeneity on serine and findserine protein kinase activity of the IPS encoding IP3R (12). receptor toward a specific peptide substrate. Several IP3R can be phosphorylated by at least three different independent purification procedures do not separate protein kinases, including PKA (18, 19), PKC (19), and calthe IP3 receptor protein from the phosphorylating ac- cium/calmodulin-dependent protein kinase I1 (CamK-11) tivity, andmany different protein kinase activators (19). These enzymes phosphorylate three distinct serines as and inhibitors do not identify protein kinases as con- evidenced by phosphopeptide mapping and phosphoamino taminants. Also, renaturation experiments reveal auacid determination (19). PKA can phosphorylate two serine tophosphorylation of the monomeric receptor on polysties, amino acids 1588 and 1755 (20), in the predicted amino vinylidene difluoride membranes. acids sequence from the mouse (9). The cerebellar (neuronal) form of the IP3R (containing the 40-amino acid insert) is preferentially phosphorylated on amino acid 1755, whereas Many cells respond to hormones and neurotransmitters the vas deferens (non-neuronal) form of the IP3R is phosthrough G-protein-dependent activation of phospholipase C, phorylated on amino acid 1588(16). Phosphorylation by PKA producing two second messengers, diacylglycerol and inositol in cerebellar membranes is associated with an alteration in 1,4,5-trisphosphate (IPS)’ (1, 2). Although diacylglycerol ac- the potency of IP3in releasing calcium (18)and may increase tivates protein kinase C (PKC) by reducing its requirement the sensitivity of the calcium stores to IP, in hepatocytes (21). The predicted amino acid sequence of IP,R (7, 9, 11) has for calcium (3), IP3 stimulates the release of internal stores of calcium (1, 2, 4), activating numerous calcium-dependent striking similarities to the calcium release channel of the processes. IP3 releases calcium by binding to a receptor ( 5 ) , sarcoplasmic reticulum, the ryanodine receptor of skeletal localized to portions of the endoplasmic reticulum (6, 7). The and cardiac muscle (22). Moreover, ATP (23-28) and calcium IP3 receptor (IP3R) hasbeen isolated (8), molecularly cloned (29-31) regulate both of these calcium release channels. ATP from mouse (9, lo), rat (ll),and human (12), and reconsti- enhances the ability of IP, to stimulate calcium fluxin reconstituted lipid vesicles (26) and increases the open probability tuted from purified components into lipid vesicleswhich of IP3-regulated calcium channels (27, 28). ATP binds satur* This work wassupported by United States Public Health Service ably to purified IP3R (26) at sites that are demonstrable with Grant MH-18501, Research Scientist Award DA-00074 (to S. H. S.), photoaffinity analogs (28). The IP3R contains at least two Training GrantGM-07309 (to D. S. B.), Predoctoral Fellowship MH- possible consensus sequences for ATP binding, with the 4010018 (to C. D. F.), and grants from the International Life Sciences amino acid splice variant generating a third consensus seInstitute and Bristol-Myers-Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This quence ( 5 ) .These recognition sites may regulate IPS-induced article must therefore be hereby marked “advertisement” in accord- calcium release by ATP, although such ATP recognition sites ance with 18 U.S.C. Section 1734 solelyto indicate this fact. occur in virtually all characterized (32) protein kinases. **To whom all correspondence and reprint requests should be Hormone receptors with tyrosine protein kinase activity addressed. are themselves autophosphorylated on tyrosines (32). To our The abbreviations used are: IP3, inositol 1,4,54risphosphate; CamK-11, calcium/calmodulin-dependentprotein kinase 11; CHAPS, knowledge no ion channelsare known to display protein 3-[(3-cholamidopropyl)~methylammonio]-l-propanesulfona~; conA, kinase-like activity, nor have receptor proteins or ion channel concanavalin A; FPLC, fast protein liquid chromatography; IPSR, complexes evidenced serine autophosphorylation activity. In inositol 1,4,5-trisphosphate receptor; PKA, protein kinase A; PKC, the present study we demonstrate that purified and reconstiprotein kinase C; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CHAPS, tuted IP3Rautophosphorylates on serine and displays protein 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid; kinase-like activity as evidenced by phosphorylation of an EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid. artificial peptide substrate.
7036
IP3 Receptor Autophosphorylation EXPERIMENTAL PROCEDURES
Materi~ls--[r-~*P]ATP and formula 963 scintillation cocktail were obtained from Du Pont-New England Nuclear. Inositol phosphates (1,4,5-IP3, 1,3,4,5-IP4, and IP,)were purchased from CalBiochem. Phospholipids for reconstitution were obtained from Avanti Polar Lipids (Birmingham, AL). Cellulose thin layer chromatography plates were purchased from Kodak. PVDF (Immobilon) was obtained from Millipore (Bedford, MA). Concanavalin A-Sepharose (ConA-Sepharose) was obtained from Pharmacia LKB Biotechnology Inc. All synthetic peptideswere obtained from Peninsula (Belmont, CA), with the exception of kemptide which was from Sigma. Heparin-agarose, CHAPS, and allother reagentswere from Sigma. PKA (33) and PKC (34) were purified from bovine heart and rat brain, respectively, as described previously (33, 34). Purification and Reconstitution of ZPa"IP3R was purified from rat cerebellum (adult male, Sprague-Dawley) using either heparin agarose and ConA-Sepharose (8, 13)or heparin-agarose and IP3 affinity chromatography (35) as described. Purity was assessed by silver staining of gels following SDS-PAGE (36). Following purification to apparent homogeneity, reconstitution of IPBRwas carried out as described (13). Autophosphorylation of ZPa-For autophosphorylation experiments either detergent-solubilized purified IP3R or purified and reconstituted IP3R was used for substrate as indicated in the figure legends and in the text. Routine incubations were for 18 h at 4 "C or 60 min at 37 "C as indicated. For routine measurements IP3R (10 pg/ ml) was incubated in a final volume of 100 pl with final concentrations of 10 mMMgC12 and 100 p~ [32P]ATP(1000 cpm/pmol ATP). PKA and PKC phosphorylations of IP3R were assayed under identical conditions except for the addition of 0.1 or 0.5 pg of phosphorylating enzyme, respectively. Incorporation of 32Pinto the IP3R was quantified by liquid scintillation spectrometry following SDS-PAGE (36). Phosphopeptide Mapping and Phosphoamino Acid DeterminationTwo-dimensional phosphopeptide maps were produced as described (37). Following phosphorylation and SDS-PAGE, IP3R containing gel pieces were digested in 1ml with 0.3 mgof thermolysin overnight a t 30 "C and lyophilized. The peptides were resuspended, spotted on cellulose thin layer plates, subjected to electrophoresis along the x axis and ascending chromatography along the y axis. The plates were then dried and autoradiographed (48h, -70 "C). For phosphoamino acid analysis (37) gel pieces were digested with thermolysin followed by acid hydrolysis in 6 M HCl for 1h at 105 "C. This hydrolysate was electrophoresed on cellulose plates at pH 1.9 followed bypH 3.5 along with phosphotyrosine, phosphoserine, and phosphothreonine standards (10 pg of each). The cellulose plates were stained with ninhydrin to detect the phosphoamino acid standard and then autoradiographed (48 h, -70 "C) to identify the migration of the 32P-labeled amino acids. Superose-6 Gel Filtration Chromatography of Purified ZP$-IP3R was purified as described (8, 13) using heparin-agarose and ConASepharose. Purified IP3R was diluted and concentrated twice using an Amicon Centriprep-30 concentrator to reduce the concentration of a-methyl mannoside to approximately 2 mM. This preparation, 100 r l (10 pg), was injected onto a Superose-6 fast protein liquid chromatography gel filtration column (Pharmacia) previously calibrated with known molecular weight standards. The column was run a t a flow rate of 0.2 ml/min with 50 mM Tris (pH 7.4, 25 T!), 1 mM EDTA, and 1 mM 8-mercaptoethanol, 1%CHAPS, and 0.6-ml fractions were collected. The column eluate was monitored by absorbance a t 280 nm, and aliquots were analyzed by autophosphorylating activity. Renaturation of ZPa Autophosphorylcrtion Activity-IP3R waspurified using either ConA-Sepharose or IP3 affinity chromatography as described (8, 35). Following biochemical isolation, purified IP3R was subjected to SDS-PAGE under standard conditions (36). After SDS-PAGE, proteins were transferred to PVDF membranes, renatured, and autophosphorylated (38), whereupon PVDF membranes were washed with KOH and water (38) and subjected to autoradiography (24 h, -70 "C). Phosphorylation of Peptide Substrates-For phosphorylation of peptide substrates, peptides (100 pg/ml) were incubated in 50 plwith final concentrations of 10 mM MgCl,, 50 p~ [3ZP]ATP(1,000 cpm/ pmol) in the presence or absence of IP3R, PKA, or PKC. Following incubation for 30 min a t 30 'C, reactions were stopped by the addition of excess EDTA, and themixtures were spotted onto P81 (Whatman, Hillsboro, OR) filters and washed 3 X 10 min in formic acid (8 ml/ liter). Since the peptides adhere to the filters a t acidic pH, specific
7037
incorporation of 32Pwas monitored by Cerenkov counting of the washed filters. RESULTS
Autophsphorylution of I P a on Serine-In initial experiments, IP3R preparations employed for autophosphorylation were purified by heparin-agarose and ConA-Sepharose chromatography providing apparently homogeneous IP3R protein, displaying a single 260-kDa band following SDS-PAGE analysis and silver staining. In all experiments IP3R preparations employed for phosphorylation studies have been first evaluated for purity by SDS-PAGE analysis and silver staining. Previously, phosphorylation experiments employed IP3R reconstituted into liposomes as described (13), because more robust phosphorylation by PKA, PKC, and CamK-I1 is obtained under these conditions (19). Thus, with the receptor reconstituted intoliposomes, PKC andCamK-I1 provide stoichiometric phosphorylation, whereas with unreconstituted detergent-solubilized IP3R, these two enzymes provide only %o as much phosphorylation. In preliminary experiments with IP3R not reconstituted into liposomes, autophosphorylation levels are about one-third of those in reconstituted liposomes (data notshown). Using reconstituted IP3R proteoliposomes, incubation at 4 "Cwith [32P]ATP andmagnesium provides substantial incorporation of radiolabeled phosphate into IP3R protein analyzed bySDS-PAGE (Fig. l).No incorporation of 32Pinto the IP3R is observed in the absence of free magnesium. Incorporation of radioactivity provides near stoichiometric levels of phosphorylation with about 0.4 mol of phosphate/mol of IP3R by 16-20 hr (Fig. 1).Incorporation of phosphate is linear for the first 4 h with a plateau between 8 and 12 h at about 70% of maximal phosphorylation, and a second small increase in levels of phosphorylation between 12 and 16 h (Fig. 1).This second increase has been observed consistently in three replications. In reconstituted vesicles we previously found that PKA phosphorylation of the IP3R in reconstituted vesicles is only 75-80% of levels in the same vesicles treated with detergent to expose receptors whose phosphorylation sites are in
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FIG. 1. Time course of autophosphorylation of reconstituted IPSRat 4 and 37 OC. IP3R was purified and reconstituted using rat cerebellum as described previously (8, 13). Reconstituted proteoliposomes were assayed for autophosphorylation as described under "Experimental Procedures." The lower stoichiometry achieved at 37 'C may deflect denaturation of the receptor protein, since IP3-activated ion channel activity also declines following incubation at 37 "C under these conditions (data not shown). Following incubation for the indicated times at either 4 and 37 "C (inset), phosphorylation reactions were stopped by the addition of 3 X sample buffer for SDSPAGE. After electrophoresis, IP3R was cut out of the gel, and the32P incorporated into the protein was determined by Cerenkov counting of the excised gel bands. For comparison, parallel samples were incubated with either the catalytic subunit of PKA or PKCas described under "Experimental Procedures." This experiment has been performed three times with essentially the same results.
Autophosphorylation IPaReceptor
7038 A
radiolabeled peptides. The peptide migrating most to the anode reflects the site labeled by PKC (19). Autophosphorylation reveals a second peptide which migrates toward the phosphothreonine anode but less than the PKCpeptide (Fig. 2B). This peptide has not been observed with any enzymatic phosphorylation phosphotyrosine of the IP3R. Purification Procedures to Substantiate the Presence of IP3R Autophosphorylation-It is conceivable that heparin/ConApurified IP3R is contaminated with a protein kinase (s) that accounts for the apparent autophosphorylation. To explore this possibility, we purified IP3R using an IPnaffinity column (35). Magnesium-dependent autophosphorylation occurs with this affinity purified preparation (Fig. 3). The time course, temperature dependence, and stoichiometry of apparent autophosphorylation are essentially the same for the IPSaffinorigin ity-purified receptor as with the ConA-purified preparation. For further purification, we subjected IP3Rpreviously purified B by heparin-agarose and ConA chromatography to Superose-6 t I a chromatography and monitored apparent autophosphoryla< ' a a tion in eluted fractions (Fig. 4). The major peak of protein 0 + emerges at afraction (No. 13) corresponding to a native 0 &a molecular weight of about lo6 daltons, consistent with our I " previous estimates for the native molecular weight of IP3R a w (8). The peak of IPsR autophosphorylation, as assayed by t 4 SDS-PAGE and autoradiography, coincides with this protein zI peak (Fig. 4). + Ferrell and Martin (38) described another technique to establish the presence of autophosphorylation. In this proce- ELECTROPHORESIS + dure proteins are purified by SDS-PAGE, transferred to niFIG. 2. Phosphoamino acid determination and two-dimen- trocellulose or PVDF, renatured, and then incubated under sional phosphopeptide mapping of autophosphorylated IP3R. phosphorylation conditions in the presence of magnesium and Phosphoamino acid analysis and phosphopeptide mapping were car- ATP. If autoradiography of the blot reveals phosphorylation, ried out as described previously (37) and under "Experimental Procedures." In A phosphoamino acids were determined following acid it cannot be attributed to an associated or contaminating hydrolysis of the thermolysin derived peptides, and all radioactivity kinase, since such a kinase would have had to display identical comigrated with the phosphoserine standard. In B phosphopeptides mobility during SDS-PAGE, conditions under which associwere mapped as described (37) following overnight digestion of a gel ated proteins dissociate unless they are covalently linked. band containing autophosphorylated IP3 receptorwith 300 pg/ml Accordingly, we subjected purified IP3R to SDS-PAGE, transthermolysin. These experiments have been replicated with essentially ferred the protein to nitrocellulose, renatured the proteins, the same results. and conducted phosphorylation and autoradiography (Fig. 5 ) . phosphoserine
the interior of the vesicles.' We suspect that the plateau in autophosphorylation followed by increased incorporation of 'lP at longer incubation times reflects the lag for ["2P]ATP and magnesium to penetrate into the liposome interior to reach receptors that possess phosphorylation sites within the liposome. Phosphorylation of IPsR by PKAandPKC is substantially more rapid than autophosphorylation, being complete by 2 h, and attainingfinal stoichiometries of 1.0 and 0.85 for PKA and PKC,respectively. To characterize the site of phosphorylation, we conducted autophosphorylation of IPsR for 24 h a t 4 "C, whereupon the receptor protein was isolated by SDS-PAGE, the IP3R band cut out of the gel, digested with thermolysin, followed by acid hydrolysis and electrophoretic separation of the amino acids (Fig. 2 A ) . PKA, PKC, and CamK-I1 phosphorylations are on serine (19) with only a small amount of threonine labeled by CamK-11. Autophosphorylation of IP,R reveals radioactivity exclusively on serine (Fig. 2 A ) . To identify peptides labeled in autophosphorylation studies, we carried out autophosphorylation and isolation of receptor protein as described above. After thermolysin treatment and lyophilization, we subjected the peptide preparation to electrophoresis in one dimension followed by chromatography in a second dimension (Fig. 2B). We detect two significantly
' C. D. Ferris and S. H. Snyder, unpublished observations.
Con A f
AP
+ -
-
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97
66
43
FIG. 3. Autoradiography of autophosphorylatedIP3R. IP3R was purified by either ConA-Sepharose (8, 13) or IP, affinity chromatography (35)and used for autophosphorylation as described under "Experimental Procedures." Following SDS-PAGE, gels were dried and exposed to film for visualization of phosphorylated proteins. The maximal stoichiometry of autophosphorylation was estimated to be between 0.3 and 0.4 mol of "'P/mol of IP,R in both preparations. C o d refers to heparin-agarose ConA-Sepharose-purified IPJR (8, 13), whereasAP refers to affinity-purified IP3Rusing heparin-agarose and IP, affinity chromatography (35). The + indicates the presence of 10 mMMgC12, whereas - indicates the absence of free magnesium in 1mM EDTA. This experiment has been replicated with essentially the same results.
Autophosphorylation IP3Receptor A
7039 TABLE I Peptides as substrates for IPSRkinase actiuity The ability of purified IPsR to phosphorylate various peptide substrates was assayed as described under“ExperimentalProcedures.” Magnesium-dependent phosphorylation of these substrates is shown as specific counts/min. The most phosphorylation occurs with kemptide, a synthetic substrate for protein kinase A. The stoichiometry of IP3R-dependent phosphorylation of kemptide is about 5% of PKA phosphorylation of kemptide underthe same conditions. This experiment was replicated with the same results.
I
FRACTION HUMDBR
Specific
Peptide CPm
1. RRLSSLRA I
,,
7
1 I O I , 12
$ 3 I, I >
FRACTION NUMDBR
FIG. 4. Autophosphorylation of purified IP3R following Superose-6 chromatography. IPsR was purified using heparin-agarose and ConA-Sepharose as described (8, 13). Purified IPSR was concentrated using Amicon centriprep 30 concentrators, and 10 pg (100 pg/ml) of purified and concentrated IP3R were applied to a Superose-6 fast protein liquid chromatography column for gel exclusionchromatography. Fractions (0.6 ml) were collected, andthe elution of protein was monitored by absorbance a t 280 nm. In A a single protein peak migrating a t approximately 1,000 kDa between 2,000 kDa)and the molecular mass standards blue dextran (VO, thyroglobulin (670 kDa) was observed. In B portions (50 pl) of each of these fractions were used to assay for autophosphorylation as described under “Experimental Procedures.” The peak of protein as indicated by absorbance a t 280 nm, and the peak of autophosphorylation occurs in fraction 13. This experiment was replicatedwith essentially the same results.
100
2. RKRSRKG 3. RRREEETEEE 4. LRRASLG (kemptide) 5. PLSRTLSVFS 6. KKRPQRATSNVS 7. RRKASGP 8. RKRSRAE
0 0 7,500 50
500 100 0
IPSR-We explored the ability of IP3R to phosphorylate synthetic peptides that areknown substrates for various protein kinases (TableI). These experimentswere carried out for two reasons: 1)to further rule out the presence of contaminating kinases by providing artificial substrates for several possible kinases and 2) to investigate the ability of IP3R to phosphorylate substrates other than IPaR itself. Substantial IP3Rdependent phosphorylation is observed only with kemptide (LRRASLG),a well characterized syntheticsubstrate for PKA. At 30 “Cphosphorylation of kemptide is linear for about Con A AP 15-20 min and plateaus at about 30 min. The extent of phosphorylation is about 5% of what we observe in parallel experiments with PKA (data notshown). Peptides not phos205phorylated by IP,R include those best characterized as substrates for casein kinase, CamK-11, and PKC. Peptide 97RRKASGP, an excellent substrate for PKA, is not appreciably phosphorylated by the IP3R. Also, the IP3R does not 66phosphorylate bovine serum albumin, bovine y-globulin, or casein. Influence of Protein Kinase Activators, Inhibitors, and Other Substances on IP3RAutophosphorylation and Phosphorylation 43- e’ of Kernptide-To further rule out the presence of contaminating kinases as well asto characterize IPsR-dependent autophosphorylation and phosphorylation of kemptide, we FIG. 5. Renaturation of IP3R autophosphorylation activity examined several known protein kinase activators and inhibfollowing SDS-PAGE and subsequent transfer to PVDF membranes. IPsR was purified using either ConA-Sepharose (ConA) or itors as well as other substances (Table 11). AutophosphoryIPS affinity chromatography ( A P ) and subjected to SDS-PAGE (ap- lation and kemptide phosphorylation by IPsR are unaffected proximately 1 pgllane) under standard conditions. The protein was by the Walsh peptide, an inhibitor of PKA, staurosporine, a transferred to a PVDF membrane and allowed to renature as de- potent inhibitor of PKC, calcium, calmodulin, cGMP, or scribed (38). Following renaturation, autophosphorylation was as- CAMP.Also, none of the inositol phosphates examined appear sayed as described (38), and the membrane was exposed to film for to regulate the IP3R phosphorylation activity. The detergents autoradiography. As positive controls, purified PKC and thecatalytic subunit of PKA were loaded together on another gel and separately CHAPS and Triton X-100 inhibit both autophosphorylation and kemptide phosphorylation. transferred to PVDF (data not shown). PKA renatured better (approximately 10-fold greater autophosphorylation per pg of enzyme) than PKC.
As positive controls for the renaturation procedure, we subjected purified PKA and PKC to the same procedure. Both PKA and PKC demonstrate autophosphorylation under these conditions with PKA providing the most intense incorporation of radiolabel (data not shown). IPaR also demonstrates selective magnesium-dependent incorporation of radiolabel, strongly implying the presence of autophosphorylation. None of the proteins analyzed (PKA, PKC, or IPaR) demonstrate any detectable autophosphorylation on PVDF membranes in the absence of free magnesium (data notshown). Phosphorylation of Peptide and Protein Substrates by the
DISCUSSION
Our major finding is that IPaRcan autophosphorylate and displays protein kinase-like activity. Contamination with other protein kinases is improbable for several reasons. First, autophosphorylation experiments have employed IP3R purified to homogeneity by two separate techniques, ConA chromatography or IPS affinity chromatography. Second, in the gel filtration chromatography studies all the phosphorylating activity is attributable to a protein of 1,000 kDa native molecular mass whose substrate is the tetrameric 260-kDa IPaR. Third, renaturation experiments with IP3R purified in two distinct procedures has ruled out a noncovalently attached contaminating protein kinase that would have been resolved
IP3Receptor Aut kophosphorylution
7040
TABLE I1 I P a autophosphorylatwn and kemptide phosphorylation: effects of protein kinase activators, inhibitors, and other substances Various substances were assayed at the indicated concentrations for their ability to effect either IP3R autophosphorylation or IP,Rdependent phosphorylation of kemptide, assayed as described under “Experimental Procedures.” These experiments were replicated with essentially the same results. ND, not determined. Control IP,R Concentration Compound Kemptide phosphorylation phosphorylation ?G
IP3 10 pM IP, 10 pM 1pS 100 p M Ca2+ 100 77pM Calmodulin 10 pM Ca2++ calmodulin cGMP CAMP Heparin 100 pg/ml EGTA 100 p M Walsh peptide 5 pM/ml Staurosporine 10 nM Triton (1%) CHAPS (1%) 27 Arachidonic acid 10 DM
88 90 95 75 88 80 88 92 95 105 94 98 30 24 96
89 92 90 94 83 91 92 100 98 100 96 25
ND
affinity. Recent evidence has suggested the possibility of autophosphorylation activity in two potassium-selective channels (41, 42). Also, the molecular cloning of a putative Ca2+-activated K’ channelhas identified a consensus sequence for ATP binding (43). If other receptors and ion channels display similar autophosphorylation and kinase activity at other substrates,those with recognition sites for ATP would seem the best candidates. REFERENCES 1. 2. 3. 4.
5. 6. 7. 8.
9. 10. 11.
by SDS-PAGE. Although a 260-kDa contaminating kinase 12. might phosphorylate IP,R in these renaturing experiments, the native molecular mass of such a contaminating kinase would have to be 1,000kDa. Two-dimensional electrophoresis 13. does not reveal any other proteins in the IPsR preparation. 14. Finally, a variety of peptide substrates, protein kinase acti- 15. vators, and inhibitorsdo not reveal any kinases contaminating the purified preparations. The demonstration that highly purified IP3R phosphorylates anothersubstrate, kemptide, 16. establishes that IP,R has a proteinkinase activity. What might be the physiological role of IP,R autophos- 17. phorylation? Autophosphorylation might alter the response of the receptor to IP,, calcium and calmedin (31, 39), and/or 18. ATP (26, 28) at its regulatory site. Since the maximal stoichiometry of autophosphorylation is about 0.4 molof 32P/mol of IP3R, it is conceivable that only a fraction of IP3Rs are 19. capable of autophosphorylation in the native state. Indeed, the recent finding of a 40-amino acid alternative splicing (16, 20. 17), which provides a new consensus sequence for nucleotide binding (5),suggests a possible explanation for this finding. Whether autophosphorylation specifically interacts with 21. phosphorylation by PKA, PKC,and CamK-I1 is unclear, 22. although we do observe autophosphorylation of the PKA and PKC sites. Also, whether any messenger molecules directly controlIP3R autophosphorylation is unknown. Autophos- 23. phorylation of the Ca2+-ATPaseof sarcoplasmic and endo24. plasmic reticulum may reflect stabilization of a phosphoenzyme intermediate (40). As with IP3R, the Ca2+-ATPaseau- 25. tophosphorylates better at 4 than at 37 “C, since the enzyme intermediate isstabilized and more easily trapped at thelower 26. temperature (40). However, Ca2+-ATPaseautophosphorylation is not absolutely magnesium-dependent, as Ca2+-ATP 27. 28. works equally well, and histidine rather than serine interacts with the phosphate group (40). To our knowledge the IP,R is the first ion channel protein 29. complex to display serine autophosphorylation activity. More- 30. over, it may be the first receptor of any sort for which serine 31. kinase activity on other substrates and on thereceptor itself has been recognized. Although IP3R does not appear to have 32. a full protein kinase domain (32), it does bind ATP with high
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