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The Journal of Immunology, 2005, 175: 6205-6210.
Copyright © 2005 by The American Association of Immunologists

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

Xiaogui Li2,*, Krishnamurthy Malathi2,*, Olga Krizanova{ddagger}, Karol Ondrias{ddagger}, Kirk Sperber§, Vitaly Ablamunits* and Thottala Jayaraman3,*,{dagger}

* Vascular Biology Laboratory, Department of Neurosurgery, St. Luke’s Roosevelt Hospital Center, New York, NY 10025; {dagger} Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032; {ddagger} Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic; and § Department of Immunobiology, Mount Sinai Medical Center, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, 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 component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The inositol 1,4,5-trisphosphate (IP3)4-gated intracellular Ca2+ release, resulting from the activation of receptor tyrosine kinases and G protein-coupled receptors, is an important regulator of various cellular processes, including proliferation and apoptosis (1, 2, 3, 4, 5, 6, 7). Several endogenous and exogenous activators of IP3R1 have been reported, including phosphorylation, positive and negative feedback by Ca2+, and association with other accessory molecules (3, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). However, the precise mechanism by which phosphorylation via specific kinases modulates IP3R function remains unknown; moreover, the significance of this modulation in the context of cell survival is unclear.

In a previous study, we reported that a cell cycle-dependent kinase, cdc2, phosphorylates IP3R1 in vitro and in vivo (3). This observation is consistent with previous reports that IP3-gated calcium release is modulated during the cell cycle (18, 19, 20). In the present study, we show that cyclins/cyclin-dependent kinases (cdks) directly interact with and modulate IP3-gated Ca2+ release via 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 regulate IP3-gated Ca2+ release during cell cycle progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

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

Cell culture and reagents

Jurkat cells (human leukemic T cell line, clone E6.1; American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% FCS and 100 U/ml penicillin and streptomycin. The cells were split every 2 days to maintain log-phase growth. Antiserum to IP3R1, raised against a synthetic peptide of the human IP3R1 sequence (aa 1829–1848), was purchased from Alexis Biochemicals. In some experiments, anti-IP3R1 was also used (a gift from G. Mignery, Loyola University, Chicago, IL) (21). The p13-Suc-1-agarose beads and mAb to cdc2 were obtained from Oncogene 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 stimulated with and without PHA for 24 h. Uninduced and PHA-induced cells were lysed in lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA, 0.1 mM NaF, 1 mM Na3VO4, 10 mM {beta}-glycerophosphate, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 0.5% Nonidet P-40 (v/v)). Immunoprecipitation was performed using these lysates and anti-IP3R1 Ab, followed by immunoblotting 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 sequences (MLKIGTS*PVKEDKEA and DPQEQVT*PVKYARL) within murine IP3R1 that contain the Ser421 and Thr799 phosphorylation residues, respectively. The polyclonal Abs were affinity purified with two cycles of purification by initially passing through nonphosphorylated peptides and then the appropriate phosphorylated peptides. The titer and specificity of the phosphospecific Abs were determined by ELISA and immunoblotting.

Western blotting, immunoprecipitation, and in vitro kinase reactions

Cell numbers were calculated, equalized across treatment groups, and lysed in ice-cold lysis buffer containing 0.5% Nonidet P-40, 25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and protease inhibitors. Cell lysates were centrifuged at 13,000 x g in a microcentrifuge, and the supernatants were subjected to immunoblotting and immunoprecipitation or incubation with Suc-1 coupled to agarose beads (22, 23). The membranes were blocked in TBST (20 mM Tris-HCl (pH 7.4), 0.9% NaCl, and 0.05% Tween 20) containing 5% nonfat dried milk for 1 h, followed by incubation with primary Abs. After extensive washing, the membranes were incubated with their respective secondary Abs (goat anti-rabbit IgG, BD Pharmingen; or goat anti-mouse IgG, Santa Cruz Biotechnology) conjugated to HRP in TBST containing 5% nonfat dried milk. The immunoblots were analyzed using the ECL detection system (Amersham). Immunoprecipitations were performed with the anti-IP3R1 Ab, as described (8), and the immune complexes were washed three times with ice-cold buffer containing 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-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leupeptin, and aprotinin (Sigma-Aldrich). The kinase assays were performed at 30°C for 10 min in 25 µl of a solution containing 50 mM Tris (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 10 µCi of [{gamma}-32P]ATP with and without exogenous cdc2/CyB. Phosphoproteins were separated by SDS-PAGE and detected by autoradiography, as described (3, 8).

Generation of wild-type and cdc2phosphorylation-deficient mutant GST proteins and pull-down assays

Generation of pGEX constructions that encode GST fusion proteins and the purification of the expressed proteins have been described previously (24). The regions corresponding to residues 375–473, 753–886, and 1–900 of mouse IP3R1 were amplified by PCR and cloned into the BamHI and EcoRI sites of pGEX2T (Amersham Biosciences). S421A and T799A mutations were introduced using the QuikChange mutagenesis kit (Stratagene), and mutations were confirmed by sequencing. GST fusion constructs 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 proteins were induced from 15 ml of cell culture (A600 = 0.5) with 0.1 mM isopropyl-{beta}-D-thiogalactopyranoside at 37°C for IP3R1/375–473 and at 12°C for IP3R1/753–886 fusion proteins. Fusion proteins were purified on glutathione-agarose beads, per the manufacturer’s instructions (Amersham Biosciences), and washed three times in PBS containing 1% Triton X-100 to remove nonspecifically bound proteins. For generating the cyclin binding-deficient IP3R1 mutants, we replaced arginine (R) in the cyclin-binding motif, RXL, with glycine (G). For the pull-down assays, wild-type and mutant 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 for 2 h. The resin was washed three times in lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Triton X-100, plus a mixture of protease inhibitors containing 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E64, bestatin, leupeptin, and aprotinin). Proteins bound to the immobilized Suc-1 were released by boiling the agarose resin in SDS-PAGE sample buffer for 5 min, followed by separation via SDS-PAGE. The proteins were transferred to nitrocellulose and immunoblotted with their respective indicated Abs.

IP3-binding assay

The IP3-binding assay was performed using the IP3R1 (1–900) fragment, as described (3). The soluble protein (30 µg) was incubated with 9.6 nM tritiated IP3 in 100 µl of binding buffer for 10 min at 4°C. The mixture was then added to 4 µl of {gamma}-globulin (50 mg/ml) and 100 µl of a solution containing 30% (w/v) polyethylene glycol 6000, 50 mM Tris-HCl (pH 8.0 at 4°C), 1 µM 2-ME, and 1 mM EDTA. After incubation at 4°C for 5 min, the protein-polyethylene glycol complex was collected by centrifugation at 10,000 x g for 5 min at 2°C. The pellets were dissolved in 180 µl of Solvable (DuPont NEN). After neutralization with 18 µl of acetic acid, the radioactivity was measured in 5 ml of Atomlight (DuPont NEN) with a liquid scintillation counter. The specific binding was calculated by subtracting the nonspecific binding (in the presence of 2 µM IP3) from the total binding measurement.

45Ca release assay

Rat brain microsomes were isolated according to Michikawa et al. (25) and then 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 mM KCl, and 2 µl of 45Ca (sp. act., 1.85 Gbq/mg; Amersham Biosciences) was then added. After incubation for 160 min on ice, the samples were adjusted to 2 mM MgCl2 and 0.2 mM Na2ATP. The samples were incubated with 60 U/ml cdc2, 60 µM roscovitine, or 5 µM digitonin for 10 min at room temperature, and were then diluted with 3 vol of 150 mM KCl, 10 mM EGTA, and 20 mM Tris-HCl (pH 7.8). Ca2+ release was induced by 1 µM IP3, and was measured after 1 min by adding 6 µl of stop solution (150 mM KCl, 10 mM EGTA, 20 mM Tris-HCl, and 1 mM La (pH 7.8)).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In a previous study, we showed that cdc2 phosphorylates IP3R1 at Ser421 and Thr799 in vitro and in vivo (3). Our sequence comparison with other IP3R revealed potential phosphorylation sites for cdc2 at Ser421 and Thr799 in IP3R1, and at Ser795 in IP3R3, with no obvious motifs in IP3R2. To test whether other IP3R are also substrates for the cdc2/CyB complex, we immunoprecipitated IP3R1, 2, and 3 from Jurkat lymphocytes with subtype-specific Abs, incubated them with the cdc2/CyB complex, and size fractionated by SDS-PAGE. To confirm the sites of phosphorylation on the IP3R, we used our phosphospecific polyclonal Abs generated against two phosphopeptides (MLKIGTS*PVKEDKEA and DPQEQVT*PVKYARL) that include the Ser421 and Thr799 phosphorylation sites (asterisks), respectively. The specificity of the affinity-purified phosphospecific Ser421 and Thr799 Abs was confirmed by dot-blot analysis using phosphorylated and nonphosphorylated peptides, as described (3). As shown in Fig. 1, A and B, the phosphospecific 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 the cdc2/CyB complex, neither of these Abs, however, detected phosphorylation of IP3R2 by cdc2/CyB. The absence of IP3R2 immunoreactivity to these Abs was not due to a paucity of IP3R2 in the immune complex (data not shown) as compared with IP3R1 (Fig. 1C). Rather, these data collectively indicate that IP3R1 and 3 are specific phosphorylation substrates of the cdk1/CyB complex and that our phosphoantibodies recognize their respective phosphorylated epitopes within IP3R1.



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  		FIGURE 1. Phosphorylation of IP3R subtypes by the cdc2/CyB complex. The IP3R subtypes were immunoprecipitated, phosphorylated, and probed 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 each panel. The phosphorylation motif Ser421 is unique to human IP3R1 (L38019) and is conserved in IP3R1 from other species. This motif is absent in human IP3R2 (D26350) and human IP3R3 (D26351). The phosphorylation motif Thr799 is present in human IP3R1 and IP3R3 (D26351), but not in IP3R2 (D26350). The numbers above, in parentheses, indicate GenBank accession numbers.

 
We next investigated whether IP3R1 binds the cdc2/CyB complex in vivo. Cell lysates from cycling Jurkat cells were adsorbed to p13-Suc-1-agarose, and we assayed the bound proteins for the presence of IP3R1 and cdc2. p13-Suc-1 binds to selected cdk with high affinity and is frequently used in affinity-ligand searches for cdc2 (22, 23). The presence of cdc2 on the p13-Suc-1-agarose was verified by immunoblotting with the anti-cdc2 Ab (Fig. 2B); moreover, the anti-IP3R1 Ab specifically recognized a protein of ~300 kDa that comigrated with IP3R1 during SDS-PAGE (Fig. 2A). Taken together, these data suggest that cdc2/CyB interacts directly or indirectly with IP3R1. To further examine the nature of the interaction between cdc2/CyB and IP3R1 during quiescent and proliferative cell stages, we analyzed immune complexes of IP3R1 from rat primary lymphocytes stimulated with and without PHA. PHA induces rapid proliferation of lymphocytes. IP3R1 immune complexes from the PHA-stimulated cells contained cdc2 and CyB, whereas the immune complexes from nonproliferating quiescent cells contained no detectable cdc2 or CyB, even though the IP3R1 level was equivalent to that in the PHA-stimulated population (Fig. 2, C–E). These results indicate that IP3R1 interacts with cdc2/CyB1 in normal primary lymphocytes after activation and that the interaction does not occur in quiescent cells due to a lack of CyB1 expression at the G0/G1 stages of the cell cycle (26). Several studies have demonstrated the presence of cyclin-binding motifs (RXL) in target proteins, thereby facilitating cyclin/cdk/target protein interactions (27, 28). Our primary sequence analysis revealed three putative CyB-binding motifs (391RHL, 441RDL, and 871RNL) in IP3R1 that are proximal to the cdc2 phosphorylation site(s). To investigate whether these sites are involved in the establishment of the CyB/cdc2/IP3R1 complex, we incubated GST fusion proteins encoding wild-type and mutant IP3R1 phosphorylation site fragments bound to glutathione-Sepharose with purified CyB and cdc2 proteins. Nonspecifically bound proteins were washed extensively, and the bound proteins were eluted, size fractionated, and probed with either anti-CyB1 or anti-cdc2 Abs in immunoblots. Although CyB1 interacted with both wild-type and phosphorylation-deficient IP3R1 fragments (375–473 and 753–886; Fig. 2F), CyB1 bound very poorly to the same IP3R1 fragments, in which three of the arginine (R) residues in the CyB-binding motif, RXL, were changed to glycine (G) (Fig. 2F). Taken together, these results suggest that CyB1 binding to IP3R1 is dependent on Arg391, Arg441, and Arg871.



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  		FIGURE 2. cdc2/CyB directly interacts with IP3R1. Lysates from cycling Jurkat cells (16 h after splitting the cells; lane 1) were mixed with p13-coupled beads. The beads were washed, and the bound proteins were eluted and resolved via SDS-PAGE, followed by immunoblotting with an Ab against IP3R1 (A) or CyB1 (B). The positive control lane (+ive) indicates lysates from Jurkat cells. Uninduced and PHA-stimulated spleen cells were lysed and immunoprecipitated with anti-IP3R1 Ab and probed with either anti-IP3R1 (C), or anti-cdc2 (D), or anti-CyB (E). The interaction between IP3R1 and CyB/cdc2 is seen only in PHA-stimulated cells. IP3R1 fusion 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 each panel.

 
A previous study showed that the IP3-binding specificity of an N-terminal IP3R1 fragment expressed in E. coli is very similar to that of the native IP3R from mouse cerebellum (29). We used a similar approach to investigate the functional effect of cdc2/CyB-mediated phosphorylation of IP3R1 on IP3 binding. For these studies, we constructed a GST fusion protein containing the first 900 N-terminal residues of IP3R1, IP3R1 (1–900), which encodes the IP3-binding pocket and the two cdc2/CyB phosphorylation sites. We induced protein expression in E. coli with isopropyl-{beta}-D-thiogalactopyranoside. Using this approach, we showed that cdc2 phosphorylation of this fusion protein increased IP3 binding by 3-fold, which was attenuated by roscovitine (3). IP3 binding to the S421A mutant was comparable to wild-type IP3R1. In contrast, the T799A mutation significantly reduced IP3 binding (p < 0.05). Mutation of both phosphorylation residue sites severely inhibited IP3 binding and is highly significant (p < 0.001) (Fig. 3A). Thus, the data in Fig. 3A show that the lack of phosphorylation at both of these sites negatively impacts IP3 binding. To determine the independent effect of cdc2/CyB-mediated phosphorylation at Ser421 and Thr799 on IP3 binding, we used wild-type and phosphorylation-deficient IP3R1 mutants (1–900), in which these potential phosphorylation sites were changed to alanine. The IP3-binding experiments were performed with increasing concentrations of unlabeled cold IP3. Specific IP3 binding is shown with wild-type (Fig. 3B), S421A (Fig. 3C), T799A (Fig. 3D), and S421A + T799A mutants (Fig. 3E). Our results show that IP3 binding to the wild type is significantly increased upon phosphorylation by cdc2/CyB complex (Fig. 3B; p < 0.05). Although S421A mutation resulted in reduced IP3 binding after phosphorylation as compared with wild-type IP3R1 (Fig. 3, B and C; p < 0.05), T799A mutation completely abrogated the effect of phosphorylation on IP3 binding (Fig. 3D; p < 0.05). Consistent with these findings, we also found that IP3 binding is severely impaired in the phosphorylation-deficient double mutant, S421A + T799A as compared with wild-type IP3R1 (Fig. 3, B and E; p < 0.001).



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  		FIGURE 3. The cdc2/CyB-mediated phosphorylation of IP3R1 increases IP3 binding. A, Protein (30 µg) from wild-type and phosphorylation-deficient IP3R mutant cells was used to determine IP3 binding, as described (3 ). The soluble protein from cells harboring an empty vector was used as a control. IP3 binding was performed with GST fusion proteins containing wild-type IP3R1 (1–900) (B), the S421A mutant (C), T799A mutant (D), or the double mutant S421A + T799A (E) using increasing concentrations of cold IP3. IP3 binding was measured in assays containing fusion 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 three independent experiments. Asterisks indicate a statistically significant difference in IP3 binding to the mutants as compared with wild type: *, p < 0.05, and **, p < 0.001.

 
We next measured IP3-induced Ca2+ release from cerebellar microsomes with and without phosphorylation to determine whether cdc2/CyB phosphorylation of IP3R1 modulates this process. The phosphorylation of IP3R1 by the cdc2/CyB complex was performed, as shown in Fig. 3. The microsomes were loaded with 45Ca, and subsequent Ca2+ release was triggered by activating IP3R with 1 µM IP3. Although the IP3-induced Ca2+ release in the control microsomes (without phosphorylation) was 34% of the total cellular Ca2+, the Ca2+ release increased by 47% upon cdc2/CyB-mediated phosphorylation (50% of the total Ca2+), which was completely blocked by roscovitine (Fig. 4).



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  		FIGURE 4. The cdc2 phosphorylation increases Ca2+ release. Microsomes from rat brain were isolated and resuspended in 10% sucrose, 1 mM 2-ME, and 10 mM MOPS/Tris (pH 7.0) (3.3 mg protein/ml), and diluted with an equal volume of KCl and with 45Ca. After incubation of the samples 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 µM roscovitine, and 5 µM digitonin for 10 min at room temperature. Ca2+ release was induced by 1 µM IP3 and measured after 1 min. The data represent the mean ± SD of three independent experiments. Each asterisk indicates a statistically significant (p < 0.05) difference in Ca2+ release in the presence or absence of the cdc2/CyB complex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our main findings are summarized as follows: 1) cdc2/CyB directly interacts with IP3R; 2) this interaction is mediated via Cy docking sites on the IP3R; 3) phosphorylation at Thr799 significantly alters IP3 binding; 4) phosphorylation increases IP3-gated Ca2+ release. Our results that Ser421 and Thr799 phosphorylation was detected in (IP3R1 immunoprecipitate) are consistent with consensus phosphorylation sites in IP3R1 sequence. Furthermore, that neither of these Abs reacted with IP3R2 immunoprecipitate suggests the specificities of the Ab reactivity (data not shown). Because the phosphospecific Abs were directed against two consensus phosphorylation motifs on IP3R1, we cannot exclude the possibility that other potential sites may be weakly phosphorylated by the cdc2/CyB complex. Nevertheless, the fact that IP3R2 contains several Ser-Pro (S-P) or Thr-Pro (T-P) residues, but lacks a consensus phosphorylation motif and reactivity with these Abs, emphasizes the utility of these reagents to monitor the phosphorylation status of consensus IP3R sites in vivo.

Previous approaches used to determine cdc2/Cy-substrate interactions include p13-Suc-1 affinity columns, immunoprecipitation, and pull-down assays (22, 23). To detect the IP3R1/cdc2/CyB complex, we used a cdc2 affinity resin, p13-Suc-1-agarose. Incubation of cell lysates with this Suc-1 resin yielded an additional protein other than cdc2 migrating at ~300 kDa that reacted with the anti-IP3R1 Ab. A lack of IP3R1 from lysates incubated with agarose beads alone suggests a specific association between IP3R1 and cdc2, although it is possible that a nonspecific interaction between IP3R1 and the Suc-1 resin could have occurred. However, this is unlikely because this association is observed only in proliferating lymphocytes after PHA stimulation and not in quiescent cells. Alternatively, lack of expression of CyB1 in quiescent cells could also have accounted for the absence of the IP3R1/CyB1/cdc2 complex (26). The data suggest the latter possibility, and we propose that CyB1 expression is critical for its association with IP3R1. Moreover, the CyB1 interaction in normal lymphocytes also shows that the finding is not unique to transformed Jurkat cells, whose growth is less dependent on growth factors.

To further determine whether IP3R interact directly with CyB1, we exploited IP3R1 fragments containing putative cyclin binding and phosphorylation sites. We mixed these fragments with purified CyB1 and assessed binding. CyB1 binding to wild-type as well as phosphorylation-deficient mutants suggests that CyB1 interacts with IP3R1 at residues distal to the phosphorylation sites. The lack of CyB1 interaction with Cy binding-deficient mutants suggests that CyB1 binding to IP3R1 is dependent on Arg391, Arg441, and Arg871. Similar interactions between cyclins and target proteins via RXL motifs have been reported (27, 28, 30). The finding that CyB1 interacts with IP3R1 suggests that it may have a role in regulating IP3R function during the cell cycle. For instance, CyB1 may target IP3R1 via a cyclin-specific interaction with its kinase partner and thereby influence the subcellular localization of phosphorylation (28). Subcellular localization may alter spatio-dynamic calcium changes and keep complexes sequestered from improper substrates, or expose the complexes to activators or inhibitors that are localized to specific compartments (31, 32).

IP3 binding is critical not only for the activation of IP3R channels, but also for their inactivation (16). The N-terminal 734 residues of IP3R1 (T734) expressed in E. coli exhibited IP3-binding characteristics similar to those of the native cerebellar IP3R. Further analyses of the N-terminal 734 residues of IP3R1 showed that a 353-residue sequence (residues 226–578) constitutes an IP3 binding region. To determine the consequences of IP3R phosphorylation at specific sites, we generated phosphorylation-deficient mutants as GST fusion proteins to elucidate the effect of phosphorylation of IP3R proteins on IP3 binding. Interestingly, significantly reduced IP3 binding was measured for the T799A mutant; this threonine residue is conserved in IP3R1 and IP3R3. Thus, this phosphorylation at Thr799 may induce a conformational change in these receptors that facilitates IP3 binding. By contrast, the S421A mutation had a relatively minimal effect. Given that Ser421 is within the IP3 binding region, this result is contrary to our expectation that Ser421 phosphorylation would modulate IP3 binding due to conformational changes. These results suggest that cdc2 phosphorylation modulates Ca2+ signaling through IP3 binding and that phosphorylation at residue Thr799 is critical for this function. Mutation analysis revealed that 10 basic residues scattered throughout this sequence are important for IP3 binding and that these residues are conserved among all members of the IP3R family (29). Of these 10 residues, three are critical, and one is known to be involved in IP3-binding specificity (33). Our results provide further understanding of the regulatory site(s) present outside of the IP3 binding region.

The biochemical mechanisms that regulate intracellular Ca2+ signals in vivo are not yet completely understood. We also investigated the effect of phosphorylation on IP3-gated Ca2+ release. Ca2+ transients occur during the G2-M phase transition and the metaphase-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 used brain microsomes because they express only IP3R1. Indeed, IP3R1-gated Ca2+ release is enhanced after phosphorylation of IP3R1 by cdc2/CyB complex. However, it is still possible that kinases other than cdc2/CyB may also modulate Ca2+ mobilization during the cell cycle. For instance, cell cycle-dependent Ca2+ changes may also be modulated via phosphorylation of IP3R by MAPK and/or Src kinases, which are active during the mitotic phase of the cell cycle. Protein phosphorylation is known to regulate numerous cellular functions, including apoptosis. Given that phosphorylation at Thr799 is important for increasing the affinity of IP3R1 for IP3, the increased phosphorylation at Thr799 after HIV infection suggests that HIV may selectively manipulate IP3-gated Ca2+ signaling.

In this study, we demonstrated that cdc2 phosphorylation alters certain IP3R properties and increases IP3-gated Ca2+ release. Given that cells deficient in IP3R fail to undergo activation-induced apoptosis (4, 6, 7) and that there is good correlation between increased cdc2/CyB activity and apoptosis in several human disorders, inappropriate and sustained phosphorylation of IP3R may result in higher cytoplasmic Ca2+ concentrations that may be detrimental to cell survival (35). This viewpoint is further supported by evidence that breast cancer resistance correlates with inactivation of cdc2/CyB activity (36, 37). Indeed, our preliminary studies indicate that phosphorylation-deficient mutant cells are relatively resistant to activation-induced apoptosis (data not shown).

The identification of apoptosis as the mechanism of cell demise/clearance under both normal physiological and pathological conditions has led to a growing interest in delineating the biochemical and molecular controls underlying this important process. For example, most self-reactive immature B cells having elevated cytoplasmic Ca2+ undergo apoptosis during development (i.e., clonal deletion) to establish immunological tolerance (38, 39, 40), whereas HIV infection causes profound immunological defects in afflicted patients, with high levels of immune activation and apoptosis of CD4+ T cells. The increased frequency of apoptosis of CD4+ T cells in HIV patients and serious perturbations of the cell cycle are associated with increased CyB1 expression and p34 cdc2 activity (41, 42, 43, 44, 45, 46, 47, 48). It is therefore likely that an abnormal relationship between T cell activation/proliferation and the occurrence of apoptosis may play a significant role in lymphocyte depletion in HIV patients. Syncytia (fusion of cells expressing the HIV-1-encoded Env gene with cells expressing the CD4/CXCR4 complex) occur upon sequential activation of CyB-cdk1, mammalian target of rapamycin, and p53; cdk1 inhibition by roscovitine/olomoucine prevents syncytial cell death elicited by HIV-1 infection of primary CD4 lymphoblasts (41). The neurotoxin protein HIV-Tat activates IP3-gated calcium stores in conferring neuronal cell death, which in turn causes AIDS-related dementia complex (42). HIV infection also causes IP3R1 to associate with the HIV-1 Nef protein, which promotes the early viral life cycle (43, 44, 45). These findings suggest the possible involvement of IP3R-mediated Ca2+ signaling in HIV pathogenesis. A detailed examination of IP3R phosphorylation pathway using blocking peptides may help to develop new strategies for treating HIV infection.

In summary, we present evidence that cdc2/CyB interacts with and phosphorylates IP3R, and that this phosphorylation increases Ca2+ release by increasing the binding affinity of IP3 for IP3R. The cdc2-mediated phosphorylation of IP3R, increased IP3 binding, and IP3R-mediated intracellular Ca2+ release are logical steps to explain the relationship between increased cdc2 activity and increased sensitivity to activation-induced apoptosis in many human disorders, including HIV. Because the cdc2/CyB complex is also necessary for the cell cycle, we suggest that other players, such as phosphatases and cy/cdk inhibitors, play an important role in regulating IP3R phosphorylation and the intracellular Ca2+ transients during the G2/M transition.


   Acknowledgments
 
We thank Peter Rappa and Lillian Medina for administrative help, and Dr. Greg Mignery for the IP3R1-specific Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
In conjunction with Columbia University, T. Jayaraman is the inventor on a patent application for "Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by cdc2". Upstate Biotechnologies, New York, has signed an agreement for marketing Abs described in the manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 pilot award from the American Cancer Society. Back

2 X.L. and K.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Thottala Jayaraman, Vascular Biology Laboratory, Department of Neurosurgery, St. Luke’s Roosevelt Hospital Center/Columbia University, New York, NY 10025. E-mail address: tj56{at}Columbia.edu Back

4 Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; cdk, cyclin-dependent kinase; CyB, cyclin B. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Berridge, M. J.. 1995. Inositol trisphosphate and calcium signaling. Ann. NY Acad. Sci. 766:31.-43. [Medline]
  2. Berridge, M. J., M. D. Bootman, P. Lipp. 1998. Calcium: a life and death signal. Nature 395:645.-648. [Medline]
  3. Malathi, K., S. Kohyama, M. Ho, D. Soghoian, X. Li, M. Silane, A. Berenstein, T. Jayaraman. 2003. Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by cdc2. J. Cell. Biochem. 90:1186.-1196. [Medline]
  4. Jayaraman, T., A. R. Marks. 1997. T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis. Mol. Cell. Biol. 17:3005.-3012. [Abstract]
  5. Jayaraman, T., A. R. Marks. 2000. Calcineurin is downstream of the inositol 1,4,5-trisphosphate receptor in the apoptotic and cell growth pathways. J. Biol. Chem. 275:6417.-6420. [Abstract/Free Full Text]
  6. Khan, A. A., M. J. Soloski, A. H. Sharp, G. Schilling, D. M. Sabatini, S. H. Li, C. A. Ross, S. H. Snyder. 1996. Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor. Science 273:503.-507. [Abstract]
  7. Sugawara, H., M. Kurosaki, M. Takata, T. Kurosaki. 1997. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 16:3078.-3088. [Medline]
  8. Jayaraman, T., E. Ondriasova, K. Ondrias, D. Harnick, A. R. Marks. 1996. Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 272:1492.-1494. [Abstract]
  9. Ferris, C. D., A. M. Cameron, D. S. Bredt, R. L. Huganir, S. H. Snyder. 1991. Inositol 1,4,5-trisphosphate receptor is phosphorylated by cyclic AMP-dependent protein kinase at serines 1755 and 1589. Biochem. Biophys. Res. Commun. 175:192.-198. [Medline]
  10. Komalavilas, P., T. M. Lincoln. 1994. Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase. J. Biol. Chem. 269:8701.-8707. [Abstract/Free Full Text]
  11. Joseph, S. K., S. V. Ryan. 1993. Phosphorylation of the inositol trisphosphate receptor in isolated rat hepatocytes. J. Biol. Chem. 268:23059.-23065. [Abstract/Free Full Text]
  12. Cameron, A. M., J. P. Steiner, D. M. Sabatini, A. I. Kaplin, L. D. Walensky, S. H. Snyder. 1995. Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. Proc. Natl. Acad. Sci. USA 92:1784.-1788. [Abstract/Free Full Text]
  13. Tu, J. C., B. Xiao, J. P. Yuan, A. A. Lanahan, K. Leoffert, M. Li, D. J. Linden, P. F. Worley. 1998. Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21:717.-726. [Medline]
  14. Bourguignon, L. Y., H. Jin, N. Lida, N. R. Brandt, S. H. Zhang. 1993. The involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2+ release from Ca2+ storage vesicles in mouse T-lymphoma cells. J. Biol. Chem. 268:7290.-7297. [Abstract/Free Full Text]
  15. Schlossmann, J., A. Ammendola, K. Ashman, X. Zong, A. Huber, G. Neubauer, G. X. Wang, H. D. Allescher, M. Korth, M. Wilm, et al 2000. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase I{beta}. Nature 404:197.-201. [Medline]
  16. Bezprozvanny, I., B. E. Ehrlich. 1994. Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca2+ channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium. J. Gen. Physiol. 104:821.-856. [Abstract/Free Full Text]
  17. Boehning, D., S. K. Joseph. 2000. Functional properties of recombinant type I and type III inositol 1,4,5-trisphosphate receptor isoforms expressed in COS-7 cells. J. Biol. Chem. 275:21492.-21499. [Abstract/Free Full Text]
  18. Deng, M. Q., S. S. Shen. 2000. A specific inhibitor of p34(cdc2)/cyclin B suppresses fertilization-induced calcium oscillations in mouse eggs. Biol. Reprod. 62:873.-878. [Abstract/Free Full Text]
  19. Tokmakov, A. A., K. I. Sato, Y. Fukami. 2001. Calcium oscillations in Xenopus egg cycling extracts. J. Cell. Biochem. 82:89.-97. [Medline]
  20. Jellerette, T., M. Kurokawa, B. Lee, C. Malcuit, S.-Y. Yoon, J. Smyth, E. Vermassen, H. De Smedt, J. B. Parys, R. A. Fissore. 2004. Cell cycle-coupled [Ca2+]i oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1. Dev. Biol. 274:94.-109. [Medline]
  21. Galvan, D. L., G. A. Mignery. 2002. Carboxy-terminal sequences critical for inositol 1,4,5-trisphosphate receptor subunit assembly. J. Biol. Chem. 277:48248.-48260. [Abstract/Free Full Text]
  22. Draetta, G., L. Brizuela, J. Potashkin, D. Beach. 1987. Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+. Cell 50:319.-325. [Medline]
  23. Dunphy, W. G., L. Brizuela, D. Beach, J. Newport. 1988. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54:423.-431. [Medline]
  24. Frangioni, J., B. G. Neel. 1993. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal. Biochem. 210:179.-187. [Medline]
  25. Michikawa, T., J. Hirota, S. Kawano, M. Hiraoka, M. Yamada, T. Furuichi, K. Mikoshiba. 1999. Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor. Neuron 23:799.-808. [Medline]
  26. Pathan, N. I., R. L. Geahlen, M. L. Harrison. 1996. The protein tyrosine kinase Lck associates with and is phosphorylated by cdc2. J. Biol. Chem. 271:27517.-27523. [Abstract/Free Full Text]
  27. Chen, J., P. Saha, S. Kornbluth, B. D. Dynlacht, A. Dutta. 1996. Cyclin-binding motifs are essential for the function of p21CIP1. Mol. Cell. Biol. 16:4673.-4682. [Abstract]
  28. Cross, F. R., M. Yuste-Rojas, S. Gray, M. D. Jacobson. 1999. Specialization and targeting of B-type cyclins. Mol. Cell 4:11.-19. [Medline]
  29. Yoshikawa, F., H. Iwasaki, T. Michikawa, T. Furuichi, K. Mikoshiba. 1999. Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor: structural and functional coupling of cleaved ligand binding and channel domains. J. Biol. Chem. 274:316.-327. [Abstract/Free Full Text]
  30. Takeda, D. Y., J. A. Wohlschlegel, A. Dutta. 2001. A bipartite substrate recognition motif for cyclin-dependent kinases. J. Biol. Chem. 276:1993.-1997. [Abstract/Free Full Text]
  31. King, R. W., R. J. Deshaies, J. M. Peters, M. W. Kirschner. 1996. How proteolysis drives the cell cycle. Science 274:1652.-1659. [Abstract/Free Full Text]
  32. Jin, P., S. Hardy, D. O. Morgan. 1998. Nuclear localization of cyclin B1 controls mitotic entry after DNA damage. J. Cell Biol. 18:875.-885.
  33. Yoshikawa, F., M. Morita, T. Monkawa, T. Michikawa, T. Furuichi, K. Mikoshiba. 1996. Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor. J. Biol. Chem. 271:18277.-18284. [Abstract/Free Full Text]
  34. Grynkiewicz, G., M. Poenie, R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440.-3450. [Abstract/Free Full Text]
  35. Shi, L., W. K. Nishioka, J. Thng, E. M. Bradbury, D. W. Litchfield, A. H. Greenberg. 1994. Premature p34cdc2 activation required for apoptosis. Science 263:1143.-1145. [Abstract/Free Full Text]
  36. Tan, M., T. Jing, K. H. Lan, C. L. Neal, P. Li, S. Lee, D. Fang, Y. Nagata, J. Liu, R. Arlinghaus, et al 2002. Phosphorylation on tyrosine-15 of p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance to taxol-induced apoptosis. Mol. Cell 9:993.-1004. [Medline]
  37. Konishi, Y., M. Lehtinen, N. Donovan, A. Bonni. 2002. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell 9:1005.-1016. [Medline]
  38. Schwartz, R. H.. 1989. Acquisition of immunologic self-tolerance. Cell 57:1073.-1081. [Medline]
  39. Goodnow, C. C.. 1996. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl. Acad. Sci. USA 93:2264.-2271. [Abstract/Free Full Text]
  40. Goodnow, C. C.. 1992. Transgenic mice and analysis of B-cell tolerance. Annu. Rev. Immunol. 10:489.-518. [Medline]
  41. Castedo, M., T. Roumier, J. Blanco, K. F. Ferri, J. Barretina, L. A. Tintignac, K. Andreau, J. L. Perfettini, A. Amendola, R. Nardacci, et al 2002. Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope. EMBO J. 21:4070.-4080. [Medline]
  42. Haughey, N. J., C.P. Holden, A. Nath, J. D. Geiger. 1999. Involvement of inositol 1,4,5-trisphosphate-regulated stores of intracellular calcium in calcium dysregulation and neuron cell death caused by HIV-1 protein tat. J. Neurochem. 73:1363.-1374. [Medline]
  43. Simmons, A., V. Aluvihare, A. McMichael. 2001. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14:763.-767. [Medline]
  44. Foti, M., L Cartier, V. Piguet, D. P. Lew, J. L. Carpentier, D. Trono, K. H. Krause. 1999. The HIV Nef protein alters Ca2+ signaling in myelomonocytic cells through SH3-mediated protein-protein interactions. J. Biol. Chem. 274:34765.-34772. [Abstract/Free Full Text]
  45. Manninen, A., K. Saksela. 2002. HIV-1 Nef interacts with inositol trisphosphate receptor to activate calcium signaling in T cells. J. Exp. Med. 195:1023.-1032. [Abstract/Free Full Text]
  46. Piedimonte, G., D. Corsi, M. Paiardiani. 1999. Unscheduled cyclin B expression and p34 cdc2 activation in T lymphocytes from HIV-infected patients. AIDS 13:1159.-1164. [Medline]
  47. Cannavo, G., M. Paiardini, D. Galati, B. Cervasi, M. Montroni, G. DeVico, D. Guetard, M. L. Bocchino, I. Picerno, M. Magnani, et al 2001. Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis. Blood 97:1756.-1764. [Abstract/Free Full Text]
  48. Fotedar, R., J. Flatt, S. Gupta, R. L. Margolis, P. Fitzgerald, H. Messier, A. Fotedar. 1995. Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B. Mol. Cell. Biol. 15:932.-942. [Abstract]

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