Cdc2/Cyclin B1 Interacts with and Modulates
Inositol 1,4,5-Trisphosphate Receptor (Type
1) Functions
This information is current as
of June 17, 2017.
Xiaogui Li, Krishnamurthy Malathi, Olga Krizanova, Karol
Ondrias, Kirk Sperber, Vitaly Ablamunits and Thottala
Jayaraman
J Immunol 2005; 175:6205-6210; ;
doi: 10.4049/jimmunol.175.9.6205
http://www.jimmunol.org/content/175/9/6205
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References
The Journal of Immunology
Cdc2/Cyclin B1 Interacts with and Modulates Inositol
1,4,5-Trisphosphate Receptor (Type 1) Functions1
Xiaogui Li,2* Krishnamurthy Malathi,2* Olga Krizanova,‡ Karol Ondrias,‡ Kirk Sperber,§
Vitaly Ablamunits,* and Thottala Jayaraman3*†
T
he 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 (1–7). 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 –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 –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
*Vascular Biology Laboratory, Department of Neurosurgery, St. Luke’s Roosevelt
Hospital Center, New York, NY 10025; †Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032; ‡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
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 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 GrantIn-Aid from the American Heart Association, Vascular Biology Fund, and a pilot
award 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, Vascular
Biology Laboratory, Department of Neurosurgery, St. Luke’s Roosevelt Hospital
Center/Columbia University, New York, NY 10025. E-mail address: tj56@
Columbia.edu
4
Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; cdk, cyclin-dependent kinase; CyB, cyclin B.
Copyright © 2005 by The American Association of Immunologists, Inc.
results provide a novel mechanism by which cyclins/cdks regulate
IP3-gated Ca2⫹ release during cell cycle progression.
Materials and Methods
Animals
Rats were obtained from Sprague-Dawley and maintained in a pathogenfree 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 SigmaAldrich. 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 ␤-glycerophosphate, 1 mM
DTT, 0.5 mM PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 10 ␮g/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.
0022-1767/05/$02.00
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The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the
importance of IP3-gated calcium (Ca2ⴙ) release to cellular apoptosis. However, the specific upstream biochemical events leading
to IP3-gated Ca2ⴙ 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 Ca2ⴙ release. Taken
together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2ⴙ 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. The Journal of Immunology, 2005, 175: 6205– 6210.
6206
Western blotting, immunoprecipitation, and in vitro kinase
reactions
Generation of wild-type and cdc2 phosphorylation-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-␤-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-9purified 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.
45
Ca 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/TrisHCl (pH 7.0) (3.3 mg protein/ml), diluted with an equal volume of 300 mM
KCl, and 2 ␮l 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 ␮M roscovitine, or 5 ␮M 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 ␮M
IP3, and was measured after 1 min by adding 6 ␮l of stop solution (150
mM KCl, 10 mM EGTA, 20 mM Tris-HCl, and 1 mM La (pH 7.8)).
Results
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 DPQEQ
VT*PVKYARL) that include the Ser421 and Thr799 phosphorylation sites (asterisks), respectively. The specificity of the affinitypurified 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/CyBphosphorylated IP3R1 in immunoblot analysis. Although the antiThr799 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.
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
IP3-binding assay
The IP3-binding assay was performed using the IP3R1 (1–900) fragment, as
described (3). The soluble protein (30 ␮g) was incubated with 9.6 nM
tritiated IP3 in 100 ␮l of binding buffer for 10 min at 4°C. The mixture was
then added to 4 ␮l of ␥-globulin (50 mg/ml) and 100 ␮l of a solution
containing 30% (w/v) polyethylene glycol 6000, 50 mM Tris-HCl (pH 8.0
at 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 at
10,000 ⫻ g for 5 min at 2°C. The pellets were dissolved in 180 ␮l of
Solvable (DuPont NEN). After neutralization with 18 ␮l 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 ␮M IP3) from the total
binding measurement.
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.
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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 ⫻ 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 ␮l of a solution containing 50 mM Tris (pH 7.4), 10 mM
MgCl2, 1 mM DTT, and 10 ␮Ci of [␥-32P]ATP with and without exogenous cdc2/CyB. Phosphoproteins were separated by SDS-PAGE and detected by autoradiography, as described (3, 8).
LINK BETWEEN CELL CYCLE AND APOPTOSIS
The Journal of Immunology
6207
that of the native IP3R from mouse cerebellum (29). We used a
similar approach to investigate the functional effect of cdc2/CyBmediated 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-␤-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 IP3binding 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).
We next measured IP3-induced Ca2⫹ release from cerebellar
microsomes with and without phosphorylation to determine
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 glutathioneSepharose 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.
FIGURE 3. The cdc2/CyB-mediated phosphorylation of IP3R1 increases IP3 binding. A, Protein (30 ␮g) 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: ⴱ, p ⬍
0.05, and ⴱⴱ, p ⬍ 0.001.
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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
871
RNL) 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 CyBbinding 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.
A previous study showed that the IP3-binding specificity of an
N-terminal IP3R1 fragment expressed in E. coli is very similar to
6208
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
45
Ca, and subsequent Ca2⫹ release was triggered by activating
IP3R with 1 ␮M 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).
Discussion
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 ␮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 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.
tween 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
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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 be-
LINK BETWEEN CELL CYCLE AND APOPTOSIS
The Journal of Immunology
Acknowledgments
We thank Peter Rappa and Lillian Medina for administrative help, and
Dr. Greg Mignery for the IP3R1-specific Ab.
Disclosures
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.
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LINK BETWEEN CELL CYCLE AND APOPTOSIS
The Journal of Immunology
CORRECTIONS
Joshi, 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 expression
and 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 online
version, which now differs from the print version as originally published.
Copyright © 2005 by The American Association of Immunologists, Inc.
0022-1767/05/$02.00
8440
CORRECTIONS
Li, X., K. Malathi, O. Krizanova, K. Ondrias, K. Sperber, V. Ablamunits, and T. Jayaraman. 2005. Cdc2/cyclin B1
interacts 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 Thottala
Jayaraman
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 HLAB*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,† Howard
Grey,* Jonathan W. Yewdell,§ and Alessandro Sette2*
*La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; †Tumor Immunology Unit, Weatherall Institute
of 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 and
Infectious 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 deficiency
confers 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 adenovirus-containing HO-1 cDNA, the source for adenoviral HO-1 cDNA was incorrectly attributed. The source is stated in
the 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, New
Orleans, LA) (29) or adenoviral ␤-galactosidase (Ad-LacZ) (BD Biosciences) were administered intranasally to each
mouse 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 administration
of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103:
1047–1054.
The Journal of Immunology
Gays, F., K. Martin, R. Kenefeck, J. G. Aust, and C. G. Brooks. 2005. Multiple cytokines regulate the NK gene complexencoded 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 shown
below.
FIGURE 1. Specificity of the CM4 mAb. A, YB2 or RNK cells transfected with Ly49 constructs were stained with
medium or first layer Abs followed by AF488 goat anti-mouse Ig. Solid lines: staining by CM4. Left broken line: medium
control. 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 medium
or saturating quantities of the unlabeled Ly49 Abs shown on the y-axis. After 20 min, AF488-labeled CM4, 4D12, or HBF
Ab was added, and incubation was continued for an additional 20 min. Median fluorescence values were determined by
flow cytometry, and the percentage inhbition caused by pretreatment with each unlabeled Ab is plotted on the y-axis. The
likelihood 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 that
were performed.
In Figure 9A, the gel image labeled Ly49A is inverted. The corrected figure is shown below.
8441
8442
CORRECTIONS
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. The
corrected 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 type
should 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 type
should be human RSV A strain, not A2 strain.
The Journal of Immunology
Chen, H., Y. K. Yip, I. George, M. Tyorkin, E. Salik, and K. Sperber. 1998. Chronically HIV-1-infected monocytic cells
induce 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 effect
of Fas-FasL interactions in CD4 and CD8 T cells cocultured with 43HIV cells; and Figure 6B, showing the apoptotic
activity of fractionated supernatant from the 43HIV cell line, are inaccurate. The corrected figures are shown below.
8443
8444
CORRECTIONS
Polyak, S., H. Chen, D. Hirsch, I. George, R. Hershberg, and K. Sperber. 1997. Impaired class II expression and antigen
uptake 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.