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1) FunctionsInositol 1,4,5-Trisphosphate Receptor (Type Cdc2/Cyclin B1 Interacts with and Modulates

JayaramanOndrias, Kirk Sperber, Vitaly Ablamunits and Thottala Xiaogui Li, Krishnamurthy Malathi, Olga Krizanova, Karol

http://www.jimmunol.org/content/175/9/6205doi: 10.4049/jimmunol.175.9.6205

2005; 175:6205-6210; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/175/9/6205.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Cdc2/Cyclin B1 Interacts with and Modulates Inositol1,4,5-Trisphosphate Receptor (Type 1) Functions1

Xiaogui Li,2* Krishnamurthy Malathi,2* Olga Krizanova,‡ Karol Ondrias,‡ Kirk Sperber,§

Vitaly Ablamunits,* and Thottala Jayaraman3*†

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates theimportance of IP3-gated calcium (Ca2�) release to cellular apoptosis. However, the specific upstream biochemical events leadingto IP3-gated Ca2� release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we showthat: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation atThr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2� release. Takentogether, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2� signaling. In addition,identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle componentand an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may con-stitute a new therapy for the treatment of several human immune disorders. The Journal of Immunology, 2005, 175: 6205–6210.

T he inositol 1,4,5-trisphosphate (IP3)4-gated intracellularCa2� release, resulting from the activation of receptortyrosine kinases and G protein-coupled receptors, is an

important regulator of various cellular processes, including prolif-eration and apoptosis (1–7). Several endogenous and exogenousactivators of IP3R1 have been reported, including phosphorylation,positive and negative feedback by Ca2�, and association withother accessory molecules (3, 8–17). However, the precise mech-anism by which phosphorylation via specific kinases modulatesIP3R function remains unknown; moreover, the significance of thismodulation in the context of cell survival is unclear.

In a previous study, we reported that a cell cycle-dependentkinase, cdc2, phosphorylates IP3R1 in vitro and in vivo (3). Thisobservation is consistent with previous reports that IP3-gated cal-cium release is modulated during the cell cycle (18–20). In thepresent study, we show that cyclins/cyclin-dependent kinases(cdks) directly interact with and modulate IP3-gated Ca2� releasevia phosphorylation. In addition, we report that cyclin B1 (CyB1)interacts directly with IP3R1 through cyclin-binding motifs. These

results provide a novel mechanism by which cyclins/cdks regulateIP3-gated Ca2� release during cell cycle progression.

Materials and MethodsAnimals

Rats were obtained from Sprague-Dawley and maintained in a pathogen-free facility of the St. Luke’s Roosevelt Hospital Center. The facility isfully accredited by the American Association for Accreditation of Labo-ratory Animal Care.

Cell culture and reagents

Jurkat cells (human leukemic T cell line, clone E6.1; American Type Cul-ture Collection) were cultured in RPMI 1640 medium containing 10% FCSand 100 U/ml penicillin and streptomycin. The cells were split every 2 daysto maintain log-phase growth. Antiserum to IP3R1, raised against a syn-thetic peptide of the human IP3R1 sequence (aa 1829–1848), was pur-chased from Alexis Biochemicals. In some experiments, anti-IP3R1 wasalso used (a gift from G. Mignery, Loyola University, Chicago, IL) (21).The p13-Suc-1-agarose beads and mAb to cdc2 were obtained from On-cogene Biosciences, and the protease inhibitor mixture was from Sigma-Aldrich. The cdc2/CyB and PHA were obtained from Calbiochem.

Spleen cell preparation and stimulation

Spleen cells were harvested from Sprague-Dawley rats and were stimulatedwith and without PHA for 24 h. Uninduced and PHA-induced cells werelysed in lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mMEDTA, 0.1 mM NaF, 1 mM Na3VO4, 10 mM �-glycerophosphate, 1 mMDTT, 0.5 mM PMSF, 1 �g/ml aprotinin, 1 �g/ml leupeptin, 10 �g/mlsoybean trypsin inhibitor, and 0.5% Nonidet P-40 (v/v)). Immunoprecipi-tation was performed using these lysates and anti-IP3R1 Ab, followed byimmunoblotting with Abs against IP3R1, cdc2, and CyB, as described (8).

Generation of phosphospecific Abs to IP3R1

Polyclonal Abs were raised in rabbits against two phosphopeptide se-quences (MLKIGTS*PVKEDKEA and DPQEQVT*PVKYARL) withinmurine IP3R1 that contain the Ser421 and Thr799 phosphorylation residues,respectively. The polyclonal Abs were affinity purified with two cycles ofpurification by initially passing through nonphosphorylated peptides andthen the appropriate phosphorylated peptides. The titer and specificity ofthe phosphospecific Abs were determined by ELISA and immunoblotting.

*Vascular Biology Laboratory, Department of Neurosurgery, St. Luke’s RooseveltHospital Center, New York, NY 10025; †Department of Medicine, College of Phy-sicians and Surgeons, Columbia University, New York, NY 10032; ‡Institute of Mo-lecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, SlovakRepublic; and §Department of Immunobiology, Mount Sinai Medical Center, NewYork, NY 10029

Received for publication March 22, 2005. Accepted for publication August 12, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the New Investigator Development Award and Grant-In-Aid from the American Heart Association, Vascular Biology Fund, and a pilotaward from the American Cancer Society.2 X.L. and K.M. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Thottala Jayaraman, VascularBiology Laboratory, Department of Neurosurgery, St. Luke’s Roosevelt HospitalCenter/Columbia University, New York, NY 10025. E-mail address: tj56@Columbia.edu4 Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; cdk, cyclin-de-pendent kinase; CyB, cyclin B.

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Western blotting, immunoprecipitation, and in vitro kinasereactions

Cell numbers were calculated, equalized across treatment groups, and lysedin ice-cold lysis buffer containing 0.5% Nonidet P-40, 25 mM HEPES (pH7.4), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and protease inhibitors.Cell lysates were centrifuged at 13,000 � g in a microcentrifuge, and thesupernatants were subjected to immunoblotting and immunoprecipitationor incubation with Suc-1 coupled to agarose beads (22, 23). The mem-branes were blocked in TBST (20 mM Tris-HCl (pH 7.4), 0.9% NaCl, and0.05% Tween 20) containing 5% nonfat dried milk for 1 h, followed byincubation with primary Abs. After extensive washing, the membraneswere incubated with their respective secondary Abs (goat anti-rabbit IgG,BD Pharmingen; or goat anti-mouse IgG, Santa Cruz Biotechnology) con-jugated to HRP in TBST containing 5% nonfat dried milk. The immuno-blots were analyzed using the ECL detection system (Amersham). Immu-noprecipitations were performed with the anti-IP3R1 Ab, as described (8),and the immune complexes were washed three times with ice-cold buffercontaining 25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM Na3VO4, 0.5%Nonidet P-40, and a mixture of protease inhibitors containing 4-(2-amino-ethyl) benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leupeptin, andaprotinin (Sigma-Aldrich). The kinase assays were performed at 30°C for10 min in 25 �l of a solution containing 50 mM Tris (pH 7.4), 10 mMMgCl2, 1 mM DTT, and 10 �Ci of [�-32P]ATP with and without exoge-nous cdc2/CyB. Phosphoproteins were separated by SDS-PAGE and de-tected by autoradiography, as described (3, 8).

Generation of wild-type and cdc2 phosphorylation-deficientmutant GST proteins and pull-down assays

Generation of pGEX constructions that encode GST fusion proteins and thepurification of the expressed proteins have been described previously (24).The regions corresponding to residues 375–473, 753–886, and 1–900 ofmouse IP3R1 were amplified by PCR and cloned into the BamHI andEcoRI sites of pGEX2T (Amersham Biosciences). S421A and T799A mu-tations were introduced using the QuikChange mutagenesis kit (Strat-agene), and mutations were confirmed by sequencing. GST fusion con-structs containing residues 375–473 (IP3R1/375–473) and 753–886(IP3R1/753–886) as well as the mutant constructs (S421A and T799A)were expressed in (Escherichia coli) JM101 (Stratagene). The proteinswere induced from 15 ml of cell culture (A600 � 0.5) with 0.1 mM iso-propyl-�-D-thiogalactopyranoside at 37°C for IP3R1/375–473 and at 12°Cfor IP3R1/753–886 fusion proteins. Fusion proteins were purified on glu-tathione-agarose beads, per the manufacturer’s instructions (AmershamBiosciences), and washed three times in PBS containing 1% Triton X-100to remove nonspecifically bound proteins. For generating the cyclin bind-ing-deficient IP3R1 mutants, we replaced arginine (R) in the cyclin-bindingmotif, RXL, with glycine (G). For the pull-down assays, wild-type andmutant proteins of fragments 375–473 and 753–886 were bound to sf-9-purified CyB1, washed extensively, and resolved on 10% SDS-PAGE gels.

Binding studies with Suc-1-agarose

p13-Suc-1-agarose was incubated with cycling Jurkat cell lysates on ice for2 h. The resin was washed three times in lysis buffer (20 mM Tris-HCl (pH7.4), 1% Triton X-100, plus a mixture of protease inhibitors containing4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leu-peptin, and aprotinin). Proteins bound to the immobilized Suc-1 were re-leased by boiling the agarose resin in SDS-PAGE sample buffer for 5 min,followed by separation via SDS-PAGE. The proteins were transferred tonitrocellulose and immunoblotted with their respective indicated Abs.

IP3-binding assay

The IP3-binding assay was performed using the IP3R1 (1–900) fragment, asdescribed (3). The soluble protein (30 �g) was incubated with 9.6 nMtritiated IP3 in 100 �l of binding buffer for 10 min at 4°C. The mixture wasthen added to 4 �l of �-globulin (50 mg/ml) and 100 �l of a solutioncontaining 30% (w/v) polyethylene glycol 6000, 50 mM Tris-HCl (pH 8.0at 4°C), 1 �M 2-ME, and 1 mM EDTA. After incubation at 4°C for 5 min,the protein-polyethylene glycol complex was collected by centrifugation at10,000 � g for 5 min at 2°C. The pellets were dissolved in 180 �l ofSolvable (DuPont NEN). After neutralization with 18 �l of acetic acid, theradioactivity was measured in 5 ml of Atomlight (DuPont NEN) with aliquid scintillation counter. The specific binding was calculated by sub-tracting the nonspecific binding (in the presence of 2 �M IP3) from the totalbinding measurement.

45Ca release assay

Rat brain microsomes were isolated according to Michikawa et al. (25) andthen resuspended in 10% sucrose, 1 mM 2-ME, and 10 mM MOPS/Tris-HCl (pH 7.0) (3.3 mg protein/ml), diluted with an equal volume of 300 mMKCl, and 2 �l of 45Ca (sp. act., 1.85 Gbq/mg; Amersham Biosciences) wasthen added. After incubation for 160 min on ice, the samples were adjustedto 2 mM MgCl2 and 0.2 mM Na2ATP. The samples were incubated with60 U/ml cdc2, 60 �M roscovitine, or 5 �M digitonin for 10 min at roomtemperature, and were then diluted with 3 vol of 150 mM KCl, 10 mMEGTA, and 20 mM Tris-HCl (pH 7.8). Ca2� release was induced by 1 �MIP3, and was measured after 1 min by adding 6 �l of stop solution (150mM KCl, 10 mM EGTA, 20 mM Tris-HCl, and 1 mM La (pH 7.8)).

ResultsIn a previous study, we showed that cdc2 phosphorylates IP3R1 atSer421 and Thr799 in vitro and in vivo (3). Our sequence compar-ison with other IP3R revealed potential phosphorylation sites forcdc2 at Ser421 and Thr799 in IP3R1, and at Ser795 in IP3R3, with noobvious motifs in IP3R2. To test whether other IP3R are also sub-strates for the cdc2/CyB complex, we immunoprecipitated IP3R1,2, and 3 from Jurkat lymphocytes with subtype-specific Abs, in-cubated them with the cdc2/CyB complex, and size fractionated bySDS-PAGE. To confirm the sites of phosphorylation on the IP3R,we used our phosphospecific polyclonal Abs generated againsttwo phosphopeptides (MLKIGTS*PVKEDKEA and DPQEQVT*PVKYARL) that include the Ser421 and Thr799 phosphoryla-tion sites (asterisks), respectively. The specificity of the affinity-purified phosphospecific Ser421 and Thr799 Abs was confirmed bydot-blot analysis using phosphorylated and nonphosphorylatedpeptides, as described (3). As shown in Fig. 1, A and B, the phos-phospecific Abs, anti-Ser421 and anti-Thr799, detected cdc2/CyB-phosphorylated IP3R1 in immunoblot analysis. Although the anti-Thr799 Ab also detected IP3R3-specific phosphorylation by thecdc2/CyB complex, neither of these Abs, however, detected phos-phorylation of IP3R2 by cdc2/CyB. The absence of IP3R2 immu-noreactivity to these Abs was not due to a paucity of IP3R2 in theimmune complex (data not shown) as compared with IP3R1 (Fig.1C). Rather, these data collectively indicate that IP3R1 and 3 arespecific phosphorylation substrates of the cdk1/CyB complex andthat our phosphoantibodies recognize their respective phosphory-lated epitopes within IP3R1.

We next investigated whether IP3R1 binds the cdc2/CyB com-plex in vivo. Cell lysates from cycling Jurkat cells were adsorbedto p13-Suc-1-agarose, and we assayed the bound proteins for thepresence of IP3R1 and cdc2. p13-Suc-1 binds to selected cdk withhigh affinity and is frequently used in affinity-ligand searches forcdc2 (22, 23). The presence of cdc2 on the p13-Suc-1-agarose was

FIGURE 1. Phosphorylation of IP3R subtypes by the cdc2/CyB com-plex. The IP3R subtypes were immunoprecipitated, phosphorylated, andprobed with the phosphospecific Abs anti-Ser421 (A) and anti-Thr799 (B).One of the blots was stripped and reprobed with Abs directed against IP3R1(C). Molecular size markers (in kDa) are indicated to the right of eachpanel. The phosphorylation motif Ser421 is unique to human IP3R1(L38019) and is conserved in IP3R1 from other species. This motif isabsent in human IP3R2 (D26350) and human IP3R3 (D26351). The phos-phorylation motif Thr799 is present in human IP3R1 and IP3R3 (D26351),but not in IP3R2 (D26350). The numbers above, in parentheses, indicateGenBank accession numbers.

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verified by immunoblotting with the anti-cdc2 Ab (Fig. 2B); more-over, the anti-IP3R1 Ab specifically recognized a protein of �300kDa that comigrated with IP3R1 during SDS-PAGE (Fig. 2A).Taken together, these data suggest that cdc2/CyB interacts directlyor indirectly with IP3R1. To further examine the nature of theinteraction between cdc2/CyB and IP3R1 during quiescent andproliferative cell stages, we analyzed immune complexes of IP3R1from rat primary lymphocytes stimulated with and without PHA.PHA induces rapid proliferation of lymphocytes. IP3R1 immunecomplexes from the PHA-stimulated cells contained cdc2 andCyB, whereas the immune complexes from nonproliferating qui-escent cells contained no detectable cdc2 or CyB, even though theIP3R1 level was equivalent to that in the PHA-stimulated popula-tion (Fig. 2, C–E). These results indicate that IP3R1 interacts withcdc2/CyB1 in normal primary lymphocytes after activation andthat the interaction does not occur in quiescent cells due to a lackof CyB1 expression at the G0/G1 stages of the cell cycle (26).Several studies have demonstrated the presence of cyclin-bindingmotifs (RXL) in target proteins, thereby facilitating cyclin/cdk/target protein interactions (27, 28). Our primary sequence analysisrevealed three putative CyB-binding motifs (391RHL, 441RDL, and871RNL) in IP3R1 that are proximal to the cdc2 phosphorylationsite(s). To investigate whether these sites are involved in the es-tablishment of the CyB/cdc2/IP3R1 complex, we incubated GSTfusion proteins encoding wild-type and mutant IP3R1 phosphory-lation site fragments bound to glutathione-Sepharose with purifiedCyB and cdc2 proteins. Nonspecifically bound proteins werewashed extensively, and the bound proteins were eluted, size frac-tionated, and probed with either anti-CyB1 or anti-cdc2 Abs inimmunoblots. Although CyB1 interacted with both wild-type andphosphorylation-deficient IP3R1 fragments (375–473 and753–886; Fig. 2F), CyB1 bound very poorly to the same IP3R1fragments, in which three of the arginine (R) residues in the CyB-binding motif, RXL, were changed to glycine (G) (Fig. 2F). Takentogether, these results suggest that CyB1 binding to IP3R1 is de-pendent on Arg391, Arg441, and Arg871.

A previous study showed that the IP3-binding specificity of anN-terminal IP3R1 fragment expressed in E. coli is very similar to

that of the native IP3R from mouse cerebellum (29). We used asimilar approach to investigate the functional effect of cdc2/CyB-mediated phosphorylation of IP3R1 on IP3 binding. For these stud-ies, we constructed a GST fusion protein containing the first 900N-terminal residues of IP3R1, IP3R1 (1–900), which encodes theIP3-binding pocket and the two cdc2/CyB phosphorylation sites.We induced protein expression in E. coli with isopropyl-�-D-thio-galactopyranoside. Using this approach, we showed that cdc2phosphorylation of this fusion protein increased IP3 binding by3-fold, which was attenuated by roscovitine (3). IP3 binding to theS421A mutant was comparable to wild-type IP3R1. In contrast, theT799A mutation significantly reduced IP3 binding ( p � 0.05).Mutation of both phosphorylation residue sites severely inhibitedIP3 binding and is highly significant ( p � 0.001) (Fig. 3A). Thus,the data in Fig. 3A show that the lack of phosphorylation at bothof these sites negatively impacts IP3 binding. To determine theindependent effect of cdc2/CyB-mediated phosphorylation atSer421 and Thr799 on IP3 binding, we used wild-type and phos-phorylation-deficient IP3R1 mutants (1–900), in which these po-tential phosphorylation sites were changed to alanine. The IP3-binding experiments were performed with increasingconcentrations of unlabeled cold IP3. Specific IP3 binding is shownwith wild-type (Fig. 3B), S421A (Fig. 3C), T799A (Fig. 3D), andS421A � T799A mutants (Fig. 3E). Our results show that IP3

binding to the wild type is significantly increased upon phosphor-ylation by cdc2/CyB complex (Fig. 3B; p � 0.05). AlthoughS421A mutation resulted in reduced IP3 binding after phosphory-lation as compared with wild-type IP3R1 (Fig. 3, B and C; p �0.05), T799A mutation completely abrogated the effect of phos-phorylation on IP3 binding (Fig. 3D; p � 0.05). Consistent withthese findings, we also found that IP3 binding is severely impairedin the phosphorylation-deficient double mutant, S421A � T799Aas compared with wild-type IP3R1 (Fig. 3, B and E; p � 0.001).

We next measured IP3-induced Ca2� release from cerebellarmicrosomes with and without phosphorylation to determine

FIGURE 2. cdc2/CyB directly interacts with IP3R1. Lysates from cy-cling Jurkat cells (16 h after splitting the cells; lane 1) were mixed withp13-coupled beads. The beads were washed, and the bound proteins wereeluted and resolved via SDS-PAGE, followed by immunoblotting with anAb against IP3R1 (A) or CyB1 (B). The positive control lane (�ive) indi-cates lysates from Jurkat cells. Uninduced and PHA-stimulated spleen cellswere lysed and immunoprecipitated with anti-IP3R1 Ab and probed witheither anti-IP3R1 (C), or anti-cdc2 (D), or anti-CyB (E). The interactionbetween IP3R1 and CyB/cdc2 is seen only in PHA-stimulated cells. IP3R1fusion proteins (fragments 375–473 and 775–886) bound to glutathione-Sepharose were incubated with sf-9-purified CyB1, washed extensively,size fractionated on SDS-PAGE, and immunoblotted with anti-CyB1 Ab(F). Molecular size markers (in kDa) are indicated to the right of eachpanel.

FIGURE 3. The cdc2/CyB-mediated phosphorylation of IP3R1 in-creases IP3 binding. A, Protein (30 �g) from wild-type and phosphoryla-tion-deficient IP3R mutant cells was used to determine IP3 binding, asdescribed (3). The soluble protein from cells harboring an empty vectorwas used as a control. IP3 binding was performed with GST fusion proteinscontaining wild-type IP3R1 (1–900) (B), the S421A mutant (C), T799Amutant (D), or the double mutant S421A � T799A (E) using increasingconcentrations of cold IP3. IP3 binding was measured in assays containingfusion proteins alone (black) or fusion proteins combined with either cdc2/CyB (triangles, red line) or cdc2/CyB and the inhibitor, roscovitine (Inh,circles, green line). The data in A–E represent the mean � SD of threeindependent experiments. Asterisks indicate a statistically significant dif-ference in IP3 binding to the mutants as compared with wild type: �, p �0.05, and ��, p � 0.001.

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whether cdc2/CyB phosphorylation of IP3R1 modulates this pro-cess. The phosphorylation of IP3R1 by the cdc2/CyB complex wasperformed, as shown in Fig. 3. The microsomes were loaded with45Ca, and subsequent Ca2� release was triggered by activatingIP3R with 1 �M IP3. Although the IP3-induced Ca2� release in thecontrol microsomes (without phosphorylation) was 34% of the to-tal cellular Ca2�, the Ca2� release increased by 47% upon cdc2/CyB-mediated phosphorylation (50% of the total Ca2�), whichwas completely blocked by roscovitine (Fig. 4).

DiscussionOur main findings are summarized as follows: 1) cdc2/CyB di-rectly interacts with IP3R; 2) this interaction is mediated via Cydocking sites on the IP3R; 3) phosphorylation at Thr799 signifi-cantly alters IP3 binding; 4) phosphorylation increases IP3-gatedCa2� release. Our results that Ser421 and Thr799 phosphorylationwas detected in (IP3R1 immunoprecipitate) are consistent withconsensus phosphorylation sites in IP3R1 sequence. Furthermore,that neither of these Abs reacted with IP3R2 immunoprecipitatesuggests the specificities of the Ab reactivity (data not shown).Because the phosphospecific Abs were directed against two con-sensus phosphorylation motifs on IP3R1, we cannot exclude thepossibility that other potential sites may be weakly phosphorylatedby the cdc2/CyB complex. Nevertheless, the fact that IP3R2 con-tains several Ser-Pro (S-P) or Thr-Pro (T-P) residues, but lacks aconsensus phosphorylation motif and reactivity with these Abs,emphasizes the utility of these reagents to monitor the phosphor-ylation status of consensus IP3R sites in vivo.

Previous approaches used to determine cdc2/Cy-substrate inter-actions include p13-Suc-1 affinity columns, immunoprecipitation,and pull-down assays (22, 23). To detect the IP3R1/cdc2/CyBcomplex, we used a cdc2 affinity resin, p13-Suc-1-agarose. Incu-bation of cell lysates with this Suc-1 resin yielded an additionalprotein other than cdc2 migrating at �300 kDa that reacted withthe anti-IP3R1 Ab. A lack of IP3R1 from lysates incubated withagarose beads alone suggests a specific association between IP3R1and cdc2, although it is possible that a nonspecific interaction be-

tween IP3R1 and the Suc-1 resin could have occurred. However,this is unlikely because this association is observed only in pro-liferating lymphocytes after PHA stimulation and not in quiescentcells. Alternatively, lack of expression of CyB1 in quiescent cellscould also have accounted for the absence of the IP3R1/CyB1/cdc2complex (26). The data suggest the latter possibility, and we pro-pose that CyB1 expression is critical for its association with IP3R1.Moreover, the CyB1 interaction in normal lymphocytes also showsthat the finding is not unique to transformed Jurkat cells, whosegrowth is less dependent on growth factors.

To further determine whether IP3R interact directly with CyB1,we exploited IP3R1 fragments containing putative cyclin bindingand phosphorylation sites. We mixed these fragments with purifiedCyB1 and assessed binding. CyB1 binding to wild-type as well asphosphorylation-deficient mutants suggests that CyB1 interactswith IP3R1 at residues distal to the phosphorylation sites. The lackof CyB1 interaction with Cy binding-deficient mutants suggeststhat CyB1 binding to IP3R1 is dependent on Arg391, Arg441, andArg871. Similar interactions between cyclins and target proteins viaRXL motifs have been reported (27, 28, 30). The finding thatCyB1 interacts with IP3R1 suggests that it may have a role inregulating IP3R function during the cell cycle. For instance, CyB1may target IP3R1 via a cyclin-specific interaction with its kinasepartner and thereby influence the subcellular localization of phos-phorylation (28). Subcellular localization may alter spatio-dy-namic calcium changes and keep complexes sequestered from im-proper substrates, or expose the complexes to activators orinhibitors that are localized to specific compartments (31, 32).

IP3 binding is critical not only for the activation of IP3R chan-nels, but also for their inactivation (16). The N-terminal 734 res-idues of IP3R1 (T734) expressed in E. coli exhibited IP3-bindingcharacteristics similar to those of the native cerebellar IP3R. Fur-ther analyses of the N-terminal 734 residues of IP3R1 showed thata 353-residue sequence (residues 226–578) constitutes an IP3

binding region. To determine the consequences of IP3R phosphor-ylation at specific sites, we generated phosphorylation-deficientmutants as GST fusion proteins to elucidate the effect of phos-phorylation of IP3R proteins on IP3 binding. Interestingly, signif-icantly reduced IP3 binding was measured for the T799A mutant;this threonine residue is conserved in IP3R1 and IP3R3. Thus, thisphosphorylation at Thr799 may induce a conformational change inthese receptors that facilitates IP3 binding. By contrast, the S421Amutation had a relatively minimal effect. Given that Ser421 iswithin the IP3 binding region, this result is contrary to our expec-tation that Ser421 phosphorylation would modulate IP3 binding dueto conformational changes. These results suggest that cdc2 phos-phorylation modulates Ca2� signaling through IP3 binding andthat phosphorylation at residue Thr799 is critical for this function.Mutation analysis revealed that 10 basic residues scatteredthroughout this sequence are important for IP3 binding and thatthese residues are conserved among all members of the IP3R fam-ily (29). Of these 10 residues, three are critical, and one is knownto be involved in IP3-binding specificity (33). Our results providefurther understanding of the regulatory site(s) present outside ofthe IP3 binding region.

The biochemical mechanisms that regulate intracellular Ca2�

signals in vivo are not yet completely understood. We also inves-tigated the effect of phosphorylation on IP3-gated Ca2� release.Ca2� transients occur during the G2-M phase transition and themetaphase-anaphase boundaries of the cell cycle; moreover,CyB1/cdk activity controls the generation of sperm-triggered Ca2�

oscillations in oocytes during the cell cycle (18, 19, 34). We usedbrain microsomes because they express only IP3R1. Indeed,IP3R1-gated Ca2� release is enhanced after phosphorylation of

FIGURE 4. The cdc2 phosphorylation increases Ca2� release. Micro-somes from rat brain were isolated and resuspended in 10% sucrose, 1 mM2-ME, and 10 mM MOPS/Tris (pH 7.0) (3.3 mg protein/ml), and dilutedwith an equal volume of KCl and with 45Ca. After incubation of the sam-ples for 160 min on ice, 2 mM MgCl2 and 0.2 mM Na2ATP were added.The samples were incubated with various combinations of 60 U/ml cdc2,60 �M roscovitine, and 5 �M digitonin for 10 min at room temperature.Ca2� release was induced by 1 �M IP3 and measured after 1 min. The datarepresent the mean � SD of three independent experiments. Each asteriskindicates a statistically significant (p � 0.05) difference in Ca2� release inthe presence or absence of the cdc2/CyB complex.

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IP3R1 by cdc2/CyB complex. However, it is still possible thatkinases other than cdc2/CyB may also modulate Ca2� mobiliza-tion during the cell cycle. For instance, cell cycle-dependent Ca2�

changes may also be modulated via phosphorylation of IP3R byMAPK and/or Src kinases, which are active during the mitoticphase of the cell cycle. Protein phosphorylation is known to reg-ulate numerous cellular functions, including apoptosis. Given thatphosphorylation at Thr799 is important for increasing the affinity ofIP3R1 for IP3, the increased phosphorylation at Thr799 after HIVinfection suggests that HIV may selectively manipulate IP3-gatedCa2� signaling.

In this study, we demonstrated that cdc2 phosphorylation alterscertain IP3R properties and increases IP3-gated Ca2� release.Given that cells deficient in IP3R fail to undergo activation-in-duced apoptosis (4, 6, 7) and that there is good correlation betweenincreased cdc2/CyB activity and apoptosis in several human dis-orders, inappropriate and sustained phosphorylation of IP3R mayresult in higher cytoplasmic Ca2� concentrations that may be det-rimental to cell survival (35). This viewpoint is further supportedby evidence that breast cancer resistance correlates with inactiva-tion of cdc2/CyB activity (36, 37). Indeed, our preliminary studiesindicate that phosphorylation-deficient mutant cells are relativelyresistant to activation-induced apoptosis (data not shown).

The identification of apoptosis as the mechanism of cell demise/clearance under both normal physiological and pathological con-ditions has led to a growing interest in delineating the biochemicaland molecular controls underlying this important process. For ex-ample, most self-reactive immature B cells having elevated cyto-plasmic Ca2� undergo apoptosis during development (i.e., clonaldeletion) to establish immunological tolerance (38–40), whereasHIV infection causes profound immunological defects in afflictedpatients, with high levels of immune activation and apoptosis ofCD4� T cells. The increased frequency of apoptosis of CD4� Tcells in HIV patients and serious perturbations of the cell cycle areassociated with increased CyB1 expression and p34 cdc2 activity(41–48). It is therefore likely that an abnormal relationship be-tween T cell activation/proliferation and the occurrence of apopto-sis may play a significant role in lymphocyte depletion in HIVpatients. Syncytia (fusion of cells expressing the HIV-1-encodedEnv gene with cells expressing the CD4/CXCR4 complex) occurupon sequential activation of CyB-cdk1, mammalian target ofrapamycin, and p53; cdk1 inhibition by roscovitine/olomoucineprevents syncytial cell death elicited by HIV-1 infection of pri-mary CD4 lymphoblasts (41). The neurotoxin protein HIV-Tat ac-tivates IP3-gated calcium stores in conferring neuronal cell death,which in turn causes AIDS-related dementia complex (42). HIVinfection also causes IP3R1 to associate with the HIV-1 Nef pro-tein, which promotes the early viral life cycle (43–45). These find-ings suggest the possible involvement of IP3R-mediated Ca2� sig-naling in HIV pathogenesis. A detailed examination of IP3Rphosphorylation pathway using blocking peptides may help to de-velop new strategies for treating HIV infection.

In summary, we present evidence that cdc2/CyB interacts withand phosphorylates IP3R, and that this phosphorylation increasesCa2� release by increasing the binding affinity of IP3 for IP3R. Thecdc2-mediated phosphorylation of IP3R, increased IP3 binding,and IP3R-mediated intracellular Ca2� release are logical steps toexplain the relationship between increased cdc2 activity and in-creased sensitivity to activation-induced apoptosis in many humandisorders, including HIV. Because the cdc2/CyB complex is alsonecessary for the cell cycle, we suggest that other players, such asphosphatases and cy/cdk inhibitors, play an important role in reg-ulating IP3R phosphorylation and the intracellular Ca2� transientsduring the G2/M transition.

AcknowledgmentsWe thank Peter Rappa and Lillian Medina for administrative help, andDr. Greg Mignery for the IP3R1-specific Ab.

DisclosuresIn conjunction with Columbia University, T. Jayaraman is the inventor ona patent application for “Inositol 1,4,5-trisphosphate receptor (type 1)phosphorylation and modulation by cdc2”. Upstate Biotechnologies, NewYork, has signed an agreement for marketing Abs described in themanuscript.

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CORRECTIONSJoshi, P. C., L. Applewhite, J. D. Ritzenthaler, J. Roman, A. L. Fernandez, D. C. Eaton, L. A. S. Brown, and D. M. Guidot.2005. Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expressionand downstream signaling in the alveolar macrophage. J. Immunol. 175: 6837–6845.

In Figure 1, panel C was omitted. The corrected figure is shown below. The error has been corrected in the onlineversion, which now differs from the print version as originally published.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

Li, X., K. Malathi, O. Krizanova, K. Ondrias, K. Sperber, V. Ablamunits, and T. Jayaraman. 2005. Cdc2/cyclin B1interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions. J. Immunol. 175: 6205–6210.

In the author line, the sequence of the first two authors is reversed. The corrected author line is shown below.

Krishnamurthy Malathi, Xiaogui Li, Olga Krizanova, Karol Ondrias, Kirk Sperber, Vitaly Ablamunits, and ThottalaJayaraman

Pasquetto, V., H.-H. Bui, R. Giannino, F. Mirza, J. Sidney, C. Oseroff, D. C. Tscharke, K. Irvine, J. R. Bennink, B. Peters,S. Southwood, V. Cerundolo, H. Grey, J. W. Yewdell, and A. Sette. 2005. HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J.Immunol. 175: 5504–5515.

The fourth author’s name, Cindy Banh, was omitted. The correct list of authors and affiliations is shown below.

Valerie Pasquetto,* Huynh-Hoa Bui,* Rielle Giannino,* Cindy Banh,* Fareed Mirza,† John Sidney,* Carla Oseroff,*David C. Tscharke,§¶ Kari Irvine,§ Jack R. Bennink,§ Bjoern Peters,* Scott Southwood,‡ Vincenzo Cerundolo,† HowardGrey,* Jonathan W. Yewdell,§ and Alessandro Sette2*

*La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; †Tumor Immunology Unit, Weatherall Instituteof Molecular Medicine, Oxford University, Oxford, United Kingdom; ‡Epimmune Incorporated, San Diego, CA 92121;§Laboratory of Viral Diseases, National Institutes of Health, Bethesda, MD 20892; and ¶ Division of Immunology andInfectious Diseases, Queensland Institute of Medical Research, Herston, Queensland, Australia

Zhang, X., P. Shan, S. Qureshi, R. Homer, R. Medzhitov, P. W. Noble, and P. J. Lee. 2005. Cutting edge: TLR4 deficiencyconfers susceptibility to lethal oxidant lung injury. J. Immunol. 175: 4834–4838.

In Materials and Methods, in the first sentence under the heading Intranasal administration of recombinant adeno-virus-containing HO-1 cDNA, the source for adenoviral HO-1 cDNA was incorrectly attributed. The source is stated inthe corrected sentence below.

Mice were anesthetized with methoxyflurane, and then 5 � 108 PFU of adenoviral HO-1 (Ad-HO-1) (a gift from K.Kolls, University of Pittsburgh Medical Center, Pittsburgh, PA, and J. Alam, Alton Ochsner Medical Foundation, NewOrleans, LA) (29) or adenoviral �-galactosidase (Ad-LacZ) (BD Biosciences) were administered intranasally to eachmouse in a volume of 50 �l as described previously (12).

The authors also wish to add the reference shown below.

29. Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. K. Choi. 1999. Exogenous administrationof heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103:1047–1054.

8440 CORRECTIONS

Gays, F., K. Martin, R. Kenefeck, J. G. Aust, and C. G. Brooks. 2005. Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells. J. Immunol. 175: 2938–2947.

In Figure 1, a sentence regarding the solid and broken lines was omitted from the legend. The corrected legend is shownbelow.

FIGURE 1. Specificity of the CM4 mAb. A, YB2 or RNK cells transfected with Ly49 constructs were stained withmedium or first layer Abs followed by AF488 goat anti-mouse Ig. Solid lines: staining by CM4. Left broken line: mediumcontrol. Right broken line: staining by positive control Abs Ly49A � A1, Ly49B � 1A1, Ly49C � 4D12, Ly49D � 4E5,Ly49E � 4D12, Ly49F � HBF, Ly49G � 4G11, Ly49H � 3D10, Ly49I � YBI. B, Cross-competition between Abs.YB2 cells transfected with Ly49E (YB2-E) and RNK cells transfected with Ly49F (RNK-F) were incubated with mediumor saturating quantities of the unlabeled Ly49 Abs shown on the y-axis. After 20 min, AF488-labeled CM4, 4D12, or HBFAb was added, and incubation was continued for an additional 20 min. Median fluorescence values were determined byflow cytometry, and the percentage inhbition caused by pretreatment with each unlabeled Ab is plotted on the y-axis. Thelikelihood that the inhibition observed was due to chance variation was determined by Student’s t test (*, p � 0.05,**, p � 0.01, ***, p � 0.001). The experiments shown are representative of three similar experiments of each type thatwere performed.

In Figure 9A, the gel image labeled Ly49A is inverted. The corrected figure is shown below.

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Rakoff-Nahoum, S., H. Chen, T. Kraus, I. George, E. Oei, M. Tyorkin, E. Salik, P. Beuria, and K. Sperber. 2001.Regulation of class II expression in monocytic cells after HIV-1 infection. J. Immunol. 167: 2331–2342.

Figure 10, demonstrating intracellular trafficking of HLA-DR after the introduciton of HIV proteins, is incorrect. Thecorrected figure is shown below.

Lukacs, N. W., K. K. Tekkanat, A. Berlin, C. M. Hogaboam, A. Miller, H. Evanoff, P. Lincoln, and H. Maassab. 2001.Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J.Immunol. 167: 1060–1065.

In Materials and Methods, in the first sentence under the heading RSV infection, the designation of the virus typeshould be human RSV A strain, not A2 strain.

Tekkanat, K. K., H. F. Maassab, D. S. Cho, J. J. Lai, A. John, A. Berlin, M. H. Kaplan, and N. W. Lukacs. 2001.IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J. Immunol. 166:3542–3548.

In Materials and Methods, in the first sentence under the heading Virus and infection, the designation of the virus typeshould be human RSV A strain, not A2 strain.

8442 CORRECTIONS

Chen, H., Y. K. Yip, I. George, M. Tyorkin, E. Salik, and K. Sperber. 1998. Chronically HIV-1-infected monocytic cellsinduce apoptosis in cocultured T cells. J. Immunol. 161: 4257–4267.

Figure 3B, demonstrating the apoptotic effect of gp120 on CD4 and CD8 cells; Figure 4B, depicting the apoptotic effectof Fas-FasL interactions in CD4 and CD8 T cells cocultured with 43HIV cells; and Figure 6B, showing the apoptoticactivity of fractionated supernatant from the 43HIV cell line, are inaccurate. The corrected figures are shown below.

8443The Journal of Immunology

Polyak, S., H. Chen, D. Hirsch, I. George, R. Hershberg, and K. Sperber. 1997. Impaired class II expression and antigenuptake in monocytic cells after HIV-1 infection. J. Immunol. 159: 2177–2188.

In Figure 5, demonstrating the inability of HIV-1-infected 43 cells to present antigen to HLA-DR2 and DR4 T cells,panels A and B are the same. The corrected figure is shown below.

8444 CORRECTIONS