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J. Biol. Chem., Vol. 277, Issue 30, 27449-27467, July 26, 2002
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From the
Received for publication, December 7, 2001, and in revised form, April 8, 2002
Cyclin G2, together with cyclin G1 and cyclin I,
defines a novel cyclin family expressed in terminally differentiated
tissues including brain and muscle. Cyclin G2 expression is
up-regulated as cells undergo cell cycle arrest or apoptosis in
response to inhibitory stimuli independent of p53 (Horne, M.,
Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl,
A. (1997) J. Biol. Chem. 272, 12650-12661). We tested
the hypothesis that cyclin G2 may be a negative regulator of cell cycle
progression and found that ectopic expression of cyclin G2 induces the
formation of aberrant nuclei and cell cycle arrest in HEK293 and
Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a
detergent-resistant compartment, suggesting an association with
cytoskeletal elements. We determined that cyclin G2 and its homolog
cyclin G1 directly interact with the catalytic subunit of protein
phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nM)
phosphatase activity coprecipitates with endogenous and ectopic cyclin
G2. We found that cyclin G2 also associates with various PP2A B'
regulatory subunits, as previously shown for cyclin G1. The PP2A/A
subunit is not detectable in cyclin G2-PP2A-B'-C complexes. Notably,
cyclin G2 colocalizes with both PP2A/C and B' subunits in
detergent-resistant cellular compartments, suggesting that these
complexes form in living cells. The ability of cyclin G2 to inhibit
cell cycle progression correlates with its ability to bind PP2A/B' and
C subunits. Together, our findings suggest that cyclin G2-PP2A
complexes inhibit cell cycle progression.
Classical cyclins promote cellular proliferation. They form
complexes with specific cyclin-dependent kinases
(CDKs)1 (1, 2), thereby
enabling CDK activation. Activated CDKs trigger cell cycle transitions
through phosphorylation of specific targets such as the tumor
suppressor Rb and cytoskeletal proteins (3). Some recently identified
cyclins and CDKs do not promote cell cycle progression per
se (4). For example, the mammalian p35-CDK5 complex is essential
for the regulation of neurite outgrowth (5, 6). Mammalian cyclin H-CDK7
and cyclin C-CDK8 regulate transcription by phosphorylating the
carboxyl terminus of RNA polymerase II (7).
Cyclin G2 is an unconventional cyclin highly expressed in cells
undergoing apoptosis (8, 9). In contrast to conventional cyclins,
cyclin G2 expression is up-regulated in B cells responding to
growth-inhibitory stimuli and during antigen-induced cell cycle arrest
and apoptosis (9), but is low in proliferating B cells (9). Cyclin G2
is also strongly expressed in adult brain cerebellum, a terminally
differentiated tissue (8, 9). Cyclin G2 shows 26% amino acid identity
with cyclin A, but its closest homolog is cyclin G1 (53% amino acid
identity). Initial cloning and sequencing of cyclin G1, then called
cyclin G, predicted a protein starting at the cyclin box and lacking an
NH2-terminal region typically present in other cyclins (10,
11). Cyclin G was later found to exist in a 45-amino acid
NH2-terminally extended form (8, 12) and renamed cyclin G1.
The cyclin G1 gene was identified as a transcriptional target of the
tumor suppressor and cell cycle checkpoint regulator p53 (11, 12). As
DNA damage induces cyclin G1 mRNA expression, it may function in
cell cycle checkpoint control (11-13). The third member of this novel
family of cyclins, cyclin I, exhibits 30% amino acid identity with
cyclin G2 and G1. Cyclin G1 and I mRNAs are constitutively
expressed throughout the cell cycle (8-10, 14). All three cyclins are
expressed in tissues rich in terminally differentiated cells,
including cardiac and skeletal muscle and various brain regions (8-10,
14). The overall expression profile of the G cyclins is atypical and
not associated with promotion of cellular proliferation but rather
suggests that they act as inhibitory coordinators of the cell cycle in
some cell types and may assist in maintaining the quiescent state of differentiated cells.
Although cyclin G2 and G1 are sequence homologs, their expression is
differentially regulated during development (9), and they probably
serve unique physiological functions. In contrast to cyclin G1, the
cyclin G2 gene is not a transcriptional target of p53, and its
expression is independent of p53 (8, 9, 13, 15). The subcellular
distribution of these two homologs is also distinctive; the primarily
nuclear localization of cyclin G1
(15)2 contrasts with the
mainly cytoplasmic and less frequent nuclear expression of cyclin G2
(this report and Ref. 15). Upon receptor-activated negative signaling
in B lymphocytes, cyclin G2 transcript levels are dramatically elevated
over an extended period, whereas cyclin G1 expression is unaffected (8,
9). In lymphocytes, cyclin G1 expression is constitutive throughout the
cell cycle, whereas cyclin G2 expression oscillates, peaking in late
S/early G2 phase of the cell cycle (8, 9). Among
differentiated tissues, cyclin G2 expression is highest in cerebellum,
but cyclin G1 is most abundant in muscle, where cyclin G2 is low
(8-10).
Controlled dephosphorylation of distinct substrates is critical for
regulation of cellular division and differentiation. Protein phosphatase 2A (PP2A) is a highly conserved serine/threonine
phosphatase essential for a plethora of cellular functions including
signal transduction, translational control, endosome trafficking, and cell cycle regulation (16-20). It is the chief target of various natural toxins and viral oncogenes (16, 21-23). PP2A dephosphorylates physiological substrates of CDKs in vitro (24) and the
Rb-related protein p107 in vivo (25). Because of its effects
on the activity of the dual specificity phosphatase Cdc25, a key
regulator of the mitotic CDK p34cdc2, PP2A activity is thought
to prevent premature entry into mitosis (26, 27). The diversity of PP2A
function is paralleled by its requisite localization to distinct
subcellular sites and specific targeting to a variety of cellular
substrates, a crucial regulatory process for the proper function of
many protein kinases (28). The 36-kDa catalytic C subunit of PP2A
(PP2A/C) exists in cells in either a dimeric core complex with a 65-kDa
"scaffolding" A subunit (PP2A/A, PR65) or, more often, in a
trimeric complex containing variable regulatory B subunits bound to the
A/C dimer (16, 29, 30). The abundance of known and possible trimeric
PP2A complexes that could be formed from two C, two A, and at least 16 B subunits (encompassed by the sequence-unrelated B (PR55), B' (PR56),
and B" (PR72) families) reflects the medley of functions for this phosphatase (16, 31-35). The various regulatory B subunits direct PP2A
to distinct subcellular localizations and modulate its substrate specificity (16, 36-41). Unlike other B subunits, some B' subunits contain a classic bipartite nuclear localization signal (31, 38, 42).
Studies in yeast showed that rabbit B' isoforms can complement
mutations in the yeast B' subunit Rts1p, but Rts1p cannot complement
mutations in the B subunit Cdc55p, indicating that different regulatory
B subunits confer enzyme specificity (43). Thus, the discovery that
cyclin G1 interacts with PP2A B' subunits (44) implicates cyclin G1 in
the regulation of PP2A and contrasts it with the classical cell
cycle-promoting cyclins.
For a better understanding of cyclin G2 function, we investigated
potential binding partners of this novel cyclin. We show that cyclin G2
interacts with various PP2A B' subunits in vitro and in
living cells, where they colocalize at distinct sites. Okamoto et al. observed that cyclin G1 interacts
with two distinct B' subunits; however, the composition of cyclin
G1-PP2A/B' complexes was not further resolved (44). To understand the
function of the cyclin G2 complex, we examined whether the cyclin
G2-PP2A/B' complex contains the other two major subunits of the
prototypical PP2A trimeric A-B'-C complex. It is possible that members
of the G cyclin family sequester B' subunits away from the PP2A/A-C
dimer and thereby change the pool of distinctly targeted PP2A
holoenzyme. Alternatively, cyclin G1 or G2 may directly regulate either
the activity or localization of PP2A by forming multimeric complexes. We found that cyclin G2 and G1 cannot only associate with a variety of
B' subunits but can also directly interact with the catalytic C subunit
of PP2A. Our data indicate that this cyclin G2-C subunit association
can also occur in vivo and that association of cyclin G2
with PP2A/B' and C excludes the A subunit. Accordingly, cyclin G2 can
form catalytically active trimeric complexes with PP2A/B' and C
subunits and thus may alter PP2A targeting or substrate specificity. A
functional consequence of high cyclin G2 expression may be a change in
the balance or local concentration of distinct PP2A complexes. This
could lead to alterations in signal transduction, cell cycle
regulation, cytoskeletal networks, or mitosis. Indeed, we observe an
induction of cell cycle arrest and unusual nuclear DNA structures in
cells overexpressing cyclin G2, indicative of an aberrant
mitosis/cytokinesis. Together, our studies point to a role for cyclin
G2 in PP2A regulation and provide a mechanism through which cyclin G2
up-regulation could contribute to cell cycle inhibition.
Reagents and Antibodies--
Glutathione- and protein
G-Sepharose, horseradish peroxidase (HRP)-conjugated protein A, and ECL
detection kits were purchased from Amersham Biosciences. The mouse
anti-HA antibody was obtained from Babco (Richmond, CA), the mouse
anti-PP2A/C from Transduction Laboratories (Lexington, KY), mouse
anti-V5 from Invitrogen (Carlsbad, CA), and goat anti-lamin B from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rat monoclonal
anti-PP2A/A antibodies 6G3 and 6F9 were kindly provided by Dr. Gernot
Walter (University of California, San Diego, CA) (30), and sheep
anti-PP2A/C and rabbit anti-pan-PP2A/B' polyclonal antibodies were from
Dr. Brian E. Wadzinski (Vanderbilt University, Nashville, TN) (40, 45).
Rabbit anti-green fluorescent protein (GFP) antiserum and the DNA
binding fluorescent dye TOTO-3 were purchased from Molecular Probes,
Inc. (Eugene, OR). HRP-conjugated rabbit anti-rat and donkey anti-sheep
IgGl; fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit
and anti-mouse IgG; lissamine rhodamine sulfonyl chloride
(LRSC)-conjugated donkey anti-rabbit, anti-mouse, and anti-goat IgG
antibodies; and Cy5-conjugated donkey anti-goat IgG were from Jackson
ImmunoResearch (West Grove, PA). HRP-conjugated goat anti-mouse IgG was
from Bio-Rad and HRP-conjugated rat anti-mouse Ig Production of Cyclin G1 and G2 Fusion
Proteins--
Complementary DNA templates encoding murine cyclin G1
and G2 (8, 9) and human PP2A/C (with and without an HA tag (46)) were
amplified by PCR using oligonucleotides containing engineered endonuclease restriction sites for subcloning in frame with a 5'
nucleotide sequence encoding glutathione S-transferase (GST) or a polyhistidine tag and T7 epitope with standard methods as described (8, 9, 47, 48). All PCR fragments were cloned into the TA
cloning vector PCR.1 (Invitrogen) and confirmed by PCR sequencing with
the AmpliTaq system (PerkinElmer Life Sciences). Cyclins G1
and G2 and PP2A/C sequences were then subcloned into pGEX 4T-1
(Amersham Biosciences), and pTrcHisA (Invitrogen) and DNA sequences
were verified as above.
GST and polyhistidine (His) fusion proteins were expressed in
Escherichia coli (NovaBlue; Novagen, Madison, WI) and
purified following the manufacturer's protocols with modifications.
Induced bacterial pellets were resuspended in 50 ml of TBS (150 mM NaCl, 15 mM Tris-Cl, pH 7.4) containing 0.1 mg/ml lysozyme. After 10 min on ice, protease inhibitors (1 µg/ml
pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 200 nM phenylmethanesulfonyl fluoride), 15 mM
dithiothreitol (GST) or 10 mM Cyclin G2 Antibodies--
New Zealand White rabbits were
immunized with affinity-purified full-length cyclin G2-GST fusion
protein eluted from glutathione-Sepharose with 15 mM
glutathione in 50 mM triethanolamine, pH 11.5, and dialyzed
against TBS. Antibodies were also raised against a
multiple-antigen peptide (49) corresponding to the first 16 amino acids
of the NH2-terminal cyclin G2 sequence
(MKDLGAKHLAGGEGVQ-K-8-MAP; anti-cyclin G2-NT). Rabbits were immunized
every 3-4 weeks with 100-250 µg of the cyclin G2-GST fusion protein
or 1-2 mg of the multiple antigen peptide solubilized in PBS and
emulsified in an equal volume of Freund's complete (initial injection)
or incomplete adjuvant in multiple subcutaneous sites. Antiserum was
preabsorbed for 90 min at 4 °C with 10 mg/ml rat liver acetone
powder (Sigma; cyclin G2 mRNA is not detectable in liver), cleared
by centrifugation, incubated for 2-4 h with cyclin G1-GST cross-linked
with dimethyl pimelimidate to glutathione-Sepharose (50), and
centrifuged to remove antibodies against the GST moiety of the fusion
protein or those cross-reactive with cyclin G1. Supernatants as well as the antisera produced against the multiple-antigen peptide preadsorbed on liver extract were then affinity-purified by incubation for 4-10 h
at 4 °C with cyclin G2-GST cross-linked with dimethyl pimelimidate to glutathione-Sepharose. Antibodies were eluted with 10 ml of 100 mM glycine, pH 2.5, and the eluates were neutralized with 1 ml of 1 M Tris-Cl, pH 8, and concentrated.
Cyclin G2 Expression Constructs and Transient Transfection of
HEK293 Cells--
HEK293 fibroblasts were cultured in Dulbecco's
modified Eagle's medium (Invitrogen), supplemented with 10%
heat-inactivated fetal bovine serum (NovaTech), 2 mM
L-glutamine, 1 mM sodium pyruvate at 37 °C
in a humidified chamber with 5% CO2. Cultures were plated at 20-30% and maintained at 50-90% confluence. DNA constructs for
expression of V5 epitope-tagged cyclin G2 or cyclin G2-GFP fusion
proteins in mammalian cells were prepared by PCR amplification of
murine cyclin G2 cDNAs with modified oligonucleotide primers. The
forward and reverse primers had engineered endonuclease restriction sites (BamHI and BglII in forward primer,
XhoI and Eco47III in reverse primer). The forward
primer contains an optimal Kozak translation initiation sequence; the
reverse primer lacked the antisense sequence for an in frame stop
codon. PCR fragments were cloned into pT-Adv
(CLONTECH, Palo Alto, CA) and sequenced. A 1.1-kbp
BamHI/XhoI restriction fragment encompassing
cyclin G2 was first subcloned into pIND-V5HisC and then a larger
BamHI/PmeI fragment encompassing cyclin G2 was
shuttled into BamHI/EcoRV-digested pcDNA3
(Invitrogen) to yield pcDNA3-cycG2-V5His for the constitutive expression of V5 epitope-tagged cyclin G2 in transient transfection. To
construct a constitutive expression vector for GFP-tagged cyclin G2, a
1.1-kbp NheI/Eco47III fragment carrying cyclin G2
was isolated from the above pIND-cycG2-V5His clone and shuttled into an
NheI/Ecl136II-cut derivative of pIND
(Invitrogen), pIND-GFP, that contains the 700-bp NheI/NotI fragment encompassing the GFP coding
sequence and a 5' polylinker sequence. A 1.9-kbp
BglII/NotI fragment from this pIND-cycG2-GFP
clone containing cyclin G2 fused in frame with GFP coding sequences was
subcloned into BamHI/NotI-digested pcDNA3 (now pcDNA3-cycG2-GFP) for constitutive expression of GFP-tagged cyclin G2.
HEK293 cells were transiently transfected using a modified calcium
phosphate precipitation protocol (51) with one of the above
pcDNA3-cyclin G2 expression constructs, expression constructs for
the HA-tagged PP2A B' subunits (pCGN B' Immunoprecipitation and Affinity Chromatography of Cyclin G2 and
PP2A from Cell Lysates--
Cells were lysed with radioimmune
precipitation buffer (10% glycerol, 1% Nonidet P-40, 0.4%
deoxycholate, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA in 50 mM Tris, pH 7.4) containing
pepstatin A (1 µg/ml), leupeptin (10 µg/ml), aprotinin (20 µg/ml), and phenylmethanesulfonyl fluoride (200 nM).
Lysates were cleared of insoluble material by centrifugation. 0.05%
SDS was added before precipitation with either 5-10 µg of
affinity-purified anti-cyclin G2, 10 µg of anti-HA, 6-10 µl of
anti-PP2A/A (6F9 on protein G-Sepharose (30)), or 10 µl (~10 µg)
of sheep anti-PP2A/C (19) and 15 µl of protein A-Sepharose (Repligen,
Needham, MA) or with 15 µl of microcystin-Sepharose (Upstate
Biotechnology, Inc., Lake Placid, NY). For in vitro
interaction studies with cyclin G1- and G2-GST fusion proteins
immobilized on glutathione-Sepharose, cell lysates were added to 20 µl of the resins and mixed for 1-4 h at 4 °C. Samples were washed
with 0.1% Tween 20 and 0.5% Nonidet P-40 in TBS before extraction
with denaturing buffer, SDS-PAGE in 10-20% gradient acrylamide gels, and immunoblotting as described (53). Anti-HA immunoprecipitates of
HA-tagged PP2A/C were also extracted at 60 °C for 20 min in a
thermomixer with 40 µl of 1.5% SDS, 50 mM Tris-Cl, pH
8.0, containing 5 mM dithiothreitol and protease inhibitors
(as above). The extracts were diluted with 360 µl of TBS, protease
inhibitors, and 1% Triton X-100 and added to cyclin G1- and G2-GST
fusion proteins immobilized on glutathione-Sepharose for further
interaction studies.
Immunofluorescence Confocal Microscopy--
HEK293 and CHO cells
were seeded at 1.5 × 105 cells/35-mm well onto
a glass coverslip coated with 10 µg/ml collagen and 1 µg/ml poly-L-lysine. Cells were allowed to adhere and were grown
for 14-18 h before transfection with a ratio of 1 µg of DNA per 3 µl of the LipofectAMINE (Invitrogen) transfection reagent in 1 ml of
serum-free medium (OptiMEM; Invitrogen) according to protocols of the
manufacturer with the following modifications. The serum-free medium
was removed 3 h after transfection and replaced with the complete
medium described above; 2 h later, the medium was exchanged again.
Coverslips were removed 24-36 h after transfection and rinsed with PBS
followed immediately by fixation with 4% paraformaldehyde for 15 min
at room temperature. For removal of soluble proteins and cellular
lipids by preextraction with Triton X-100, coverslips were briefly
rinsed in the microtubule-stabilizing buffer PHEM (45 mM
PIPES, 45 mM HEPES, 10 mM EGTA, 5 mM MgCl2, pH 6.9), followed by extraction of
the cells for 90-95 s in PHEM containing 0.5% Triton X-100 and by
another rapid rinse in PHEM immediately prior to fixation essentially
as described (54). After fixation, the coverslips were rinsed in PBS
and stored at 4 °C in PBS plus 0.02% sodium azide.
Specimens were permeabilized with 0.3% Triton X-100 in PBS for 20 min
(unless preextracted; see above), incubated with blocking solution (PBS
containing 2% glycerol, 25 mM NH4Cl, 2.5%
fetal bovine serum, and 0.5% donkey serum) for 2 h at 20 °C or
overnight at 4 °C and then with primary antibodies in blocking
solution for 1.5 h at 20 °C, and then washed three times with
PBS and four times with TBS. Coverslips were incubated for 30 min at
20 °C with secondary antibodies (and 6 µM TOTO-3 when
staining for DNA) in blocking solution containing 20 mM
Tris-Cl followed by washing five times with TBS and twice with
H2O before mounting with the ProLong Antifade Kit from
Molecular Probes. Of note, control experiments were performed to
exclude the possibility of nonspecific labeling or, in the case of
double-labeling, cross-reactivity of secondary antibodies with primary
antibodies as well as bleed-through from one channel into another.
Cell Cycle Analysis by Fluorescence-activated Cell Sorting (FACS)
and Flow Cytometry--
The DNA distribution profiles of the cyclin
G2, GFP-expressing, and null cells were determined through a
combination of cell sorting and flow cytometry using a
FacstarPLUS dual laser cytometer (Becton Dickinson, San
Jose, CA). Cultures transfected with either a cyclin G2-GFP or control
GFP expression vector were harvested, washed, and resuspended at
1.5 × 106 cells/ml in media supplemented with
20 µg/ml of the cell-permeable DNA dye Hoechst 33342 and incubated
for ~45 min at 37 °C. Cell suspensions were then filtered through
a 70-µm Nitex nylon mesh (Sefar America, Inc.) and further incubated
at room temperature with 5 µg/ml of the cell-impermeable DNA dye
propidium iodide (PI) for 5 min. The cells were sorted with the
cytometer's argon I-90 laser set at 488 nm in the primary position
(GFP, G2-GFP) and the krypton laser 300 series tuned to a multiline
ultraviolet spectrum in the secondary position for Hoechst 33342 (DNA).
The GFP signal was collected through a 450/50 band pass filter. The PI
signal was excited with the argon laser and collected through a 660/22
band pass filter. Both lasers were set with 100 milliwatts of light.
Each sample was sorted, and data were collected (50,000 events) using
CellQuest acquisition and analysis software (Becton Dickinson). Final
analysis of the collected data was done using FlowJo 3.2 software (Tree
Star, Inc., San Carlos, CA). PI-positive cells and doublets were
excluded to ensure that only single viable cells were used for analysis
of DNA content and GFP expression.
BrdUrd incorporation was determined to measure relative amounts of DNA
synthesis as described (8, 9) with the following modifications. The
BrdUrd (20 µM) pulse (15 min)-labeled cells were fixed in
a PBS buffer containing 10 mM EDTA and 0.5%
paraformaldehyde for 5-10 min to retain the GFP signal and washed
again prior to fixation and permeabilization in ice-cold 70% methanol.
The incorporated BrdUrd present in the denatured DNA was detected with
an unlabeled mouse anti-BrdUrd monoclonal primary antibody (40 ng/µl;
Caltag) in conjunction with an Alexa-660-labeled goat anti-mouse
secondary antibody (1:500; Molecular Probes). The cell populations in
each transfection were measured according to the amount of green
fluorescence (G2-GFP, GFP) or no fluorescence (nonexpressing) DNA
content (PI) and BrdUrd incorporation (Alexa 660 fluorescence) by flow
cytometry using a FACSCalibur cytometer equipped with two air-cooled
lasers. 50,000 events were acquired using CellQuest software (Becton
Dickinson). The individual cells of each of these segregated
populations were then analyzed for their DNA content profile and degree
of DNA synthesis (BrdUrd incorporation) using FlowJo 3.4 analysis software.
Kinase and Phosphatase Assays--
HEK 293 and CHO cells were
transfected with pcDNA3-G2GFP, pcDNA3-G2V5, or pcDNA3-GFP
and for phosphatase assays cotransfected with pcDNA3-HAPP2A/C. For
CDK2 kinase activity assays, the G2GFP- or GFP-transfected cell
population was collected ~27-30 h post-transfection (~60%
confluence) and sorted live via an SE Vantage high speed FACS cytometer
(Becton Dickinson) into GFP-positive and negative cell populations
using an argon laser tuned to 488 nm. The cytometer was set at 35 p.s.i. with a droplet frequency of 5900 kHz. Doublets were excluded
using "time of flight" measurements. The expressing population
collection gate included cells in the upper two-thirds of GFP signal
intensity, and nonexpressor gate was limited to those under the first
decalog with the lowest GFP signal. The sorted cell populations
(~5 × 106) were kept at 2-4 °C and concentrated
by centrifugation. Cell pellets were lysed in radioimmune precipitation
buffer (supplemented with 25 mM NaF, 20 mM
For phosphatase assays, cell pellets harvested from the various
transfections or rat brain cerebellum were solubilized in modified
radioimmune precipitation buffer (without phosphatase inhibitors), and
cleared lysates containing 1.5 or 3.0 mg of protein, respectively, were
subjected to immunoprecipitation in triplicate with either rabbit
anti-cyclin G2 or control IgG. The immunocomplexes were collected by
centrifugation, washed three times with phosphatase wash buffer (150 mM NaCl, 15 mM Tris, pH 7.4, 1% Nonidet P-40, 0.4% deoxycholate, and 0.05% SDS), and washed one time with
phosphate-free double-distilled H2O, and then bound
phosphatase activity was determined by measuring the phosphate released
following incubation with a target phosphopeptide (RRApTVA; where pT
represents phosphothreonine). Amounts of released phosphate were
determined using a malachite green/molybdate-based assay following
protocols prescribed by the manufacturer (serine/threonine phosphatase
assay system; Promega) with modifications. Immunocomplexes were mixed
at room temperature in 50 µl of assay buffer (330 µM
phosphopeptide, 165 µM imidazole, pH 7.2, 660 µM EDTA, 0.066% Cellular Effects of Ectopic Cyclin G2 Expression and Its
Subcellular Distribution--
Several trials to establish stable cell
clones constitutively or inducibly expressing cyclin G2 yielded only
weak expressors rapidly lost upon expansion. Therefore, transient
transfection assays were utilized to characterize cyclin G2. All
figures shown were obtained with an anti-cyclin G2 antibody produced
against a full-length cyclin G2-GST fusion protein. The specificity of its immunoreactivity was confirmed on immunoblots of cyclin G1-GST, G2-GST, and GST alone (cyclin G2 antibodies do not cross-react with
cyclin G1; Fig. 1A,
lanes 4-6) and in parallel assays with anti-cyclin G2-NT antibodies raised against a peptide derived from
NH2-terminal sequences of cyclin G2 (data not shown).
Anti-cyclin G2 antibodies recognized a single protein band of about 48 kDa and a group of bands just below 70 kDa in total cell lysates of HEK293 cells expressing cyclin G2-V5His and G2-GFP fusion proteins, respectively (Fig. 1A, lanes 1 and
2). Apparent Mr values of the immunoreactive bands are as expected for the fusion proteins. The
detection of multiple bands migrating within the same
Mr range in lysates from cultures expressing
G2-GFP probably reflects limited proteolytic breakdown of the cyclin
G2-GFP protein. No immunoreactive bands were detectable in cell lysates
from HEK293 cells expressing GFP alone (Fig. 1, lane
3).
HEK293 and CHO cultures expressing cyclin G2-GFP and G2-V5His were
labeled with anti-cyclin G2 (red) and in the latter case anti-V5 (green) antibodies (Fig. 1). No anti-cyclin
G2 immunoreactivity was observed in cells not expressing cyclin G2
fusion proteins (see no GFP or anti-V5 signal in Fig. 1B;
nuclei detected with the DNA dye TOTO-3 pseudocolorized in
blue). Accordingly, the anti-cyclin G2 antibodies
specifically recognize the ectopically expressed cyclin G2 fusion
proteins and do not cross-react with other proteins in these cells.
Lipid-mediated transfection of cytomegalovirus-driven expression
vectors for cyclin G2-GFP and G2-V5His resulted in an expression
frequency of about 15-20% of the HEK293 and 30-60% of the CHO cell
populations 20-24 h post-transfection. In contrast to control GFP
transfectants, the number of cyclin G2-expressing cells decreased
substantially in both cell lines by 48-56 h, suggesting inhibitory
effects of ectopic cyclin G2 expression on cells. Furthermore, DNA
staining revealed an aberrant, fragmented, or multilobed appearance of
the nuclei; at 29-34 h post-transfection, ~60% of the cyclin
G2-positive cells had abnormal nuclei (see Fig. 1B). Less
than 10% of mock or empty GFP vector-transfected cells showed nuclear
abnormalities. To further characterize these nuclear abnormalities,
HEK293 cultures expressing cyclin G2 fusion proteins were immunostained
with antibodies against lamin B, a structural protein of the nuclear
envelope that is degraded by caspases in apoptotic cells. Cells
expressing cyclin G2-GFP (Fig. 1C, left
panels, green) contained nuclei with multiple
DNA-stained lobes (Fig. 1C, right
panels), each surrounded by a lamin B (Fig. 1C,
middle panels)-positive membrane, indicating an
intact nuclear envelope. This observation, together with the absence of
DNA overcondensation in the majority of these cyclin G2-overexpressing
cells (see Fig. 1, B-D), argues that the principal nuclear
aberration observed does not reflect apoptotic nuclei but rather
results from defect in a mitotic or cytokinetic process. Furthermore,
TUNEL staining did not reveal an obvious correlation of the cyclin
G2-expressing cells displaying nuclear aberrations with TUNEL-positive
apoptotic nuclei (data not shown).
Cyclin G2-GFP as well as G2-V5His is detectable throughout the
cytoplasmic region and often but not always in the nucleus. In many CHO
cells (e.g. Figs. 1D and 4D) and also
some HEK293 cells, this distribution appears rather punctate (Fig.
1C and data not shown). In addition, spots with strong
cyclin G2-GFP fluorescence or cyclin G2 immunosignals are visible in
the perinuclear region of HEK293 cells. An overlay of the red and green
channels results in a yellow color throughout most of the cyclin
G2-expressing cells. Because of the often quite pronounced punctate
distribution pattern for cyclin G2, we examined whether cyclin G2 is
associated with a Triton X-100-insoluble fraction of transfected cells.
In fact, preextraction of HEK293 cells (data not shown) and CHO cells for 90 s with Triton X-100 in the cytoskeleton-stabilizing PHEM buffer prior to fixation revealed that a large portion of cyclin G2 was
not extractable (Figs. 1D, 4, and 6E). In
contrast, other proteins such as BiP, a marker protein for the
endoplasmic reticulum and ectopically expressed GFP, which behaves as a
soluble cytosolic marker, were efficiently removed by this Triton X-100
preextraction method (data not shown). Our data indicate that cyclin G2
is to a significant extent associated with Triton X-100-insoluble
subcellular structures (e.g. nuclei and the cytoskeleton).
Cyclin G2 Expression Promotes Cell Cycle Arrest--
Our earlier
observations of up-regulation of endogenous cyclin G2 expression during
cell cycle arrest and apoptosis prompted us to examine the effect of
ectopic cyclin G2 expression on cell cycle progression in CHO and
HEK293 cells. Live cell cultures ectopically expressing cyclin G2GFP or
GFP were harvested and stained with the cell-permeant DNA dye Hoechst
33342 and the cell-impermeant DNA dye PI. Flow cytometric
analysis revealed the DNA content of the GFP-positive and PI-negative
cells (Fig. 2). DNA content profiles of
the nonexpressing (GFP-negative) fractions of both populations appeared
normal, exhibiting no appreciable difference between themselves and
nontransfected cycling cell populations (Fig. 2A and data
not shown). However, populations expressing moderate and high levels of
cyclin G2GFP revealed a strong G1 phase cell cycle arrest
by 21 h post-transfection. Each cyclin G2-positive population
showed a considerable accumulation of cells with a G1 phase
DNA content concomitant with a decrease of cells in both the S and
G2 phases. In contrast, control cells expressing GFP alone
continued to grow asynchronously with cell cycle distributions comparable with those of parental cells lacking GFP expression (Fig 2A). Similar results were observed with HEK cyclin G2
and GFP transfectants, the percentage of cyclin G2-positive
cells accumulated in the G1 phase (73%) relative to the
normal cell cycle distribution of GFP alone and parental control cells.
A similar accumulation of G1 phase cells was observed with
cells expressing untagged cyclin G2 (coexpressed with GFP) but never in
the control GFP-alone populations (see below). Comparison of the DNA
profiles of CHO and HEK cyclin G2GFP expressors with nonexpressors and
parallel GFP control populations harvested after longer times in
culture showed a clear accumulation of cells with a broad
G1/S phase distribution and an alteration of the
G2/M phase profile suggestive of aneuploidy (data not
shown).
To further dissect the cell cycle status of cyclin G2-expressing
populations, cyclin G2GFP and control GFP transfectant cultures were
pulse-labeled with BrdUrd for 15 min immediately prior to harvest (28 h
post-transfection) and analyzed for the amount of newly synthesized DNA
via measurement of BrdUrd label incorporation. GFP expression in the
control did not alter BrdUrd incorporation profiles relative to the
nonexpressing population of the same transfection (Fig. 2B)
and the nontransfected parental control (data not shown). In contrast,
the comparable population of cyclin G2GFP-positive cells exhibited very
little BrdUrd incorporation (5%), with the majority of the cells
arrested in G1 phase (Fig. 2B). Longer pulses of
45 min revealed a similar inhibition of DNA synthesis in cyclin
G2GFP-expressing cells (9% BrdUrd incorporation) compared with 31% of
the nonexpressing cells and control GFP-expressing cells (data not shown).
CDK2 kinase activity is crucial for normal transition of a cell through
the G1/S restriction point and passage through S phase. To
determine whether CDK2 activity was reduced in cyclin G2GFP-expressing CHO and HEK cells compared with GFP control cells, moderate to high
expressing cells and nonexpressing cells of each culture were collected
live from the same transfected culture via cell sorting.
Immunoprecipitations were performed in duplicate with anti-CDK2 and
control IgG from identical protein amounts (80 µg) of lysate of each
sorted population. Immunocomplexes were incubated with histone H1
substrate and [ Cyclin G2 Is Associated with PP2A B'
We investigated the potential interaction of cyclin G2 and cyclin G1
with the rabbit PP2A B'
To investigate the possible association of cyclin G2 with PP2A B'
subunits in mammalian cells, lysates were obtained from HEK293 cultures
transiently expressing cyclin G2 with and without HA-tagged B'
subunits. Anti-cyclin G2 immunocomplexes isolated from lysates of these
transfected cultures were immunoblotted with anti-HA antibodies.
Immunoreactive proteins migrating with an apparent
Mr of 62,000 and 69,000, corresponding to
HA-tagged B'
For a more quantitative analysis, analogous immunoprecipitation
experiments using anti-cyclin G2 antibodies were performed from lysates
containing 500 µg of protein followed by immunoblotting and
immunosignals compared with those obtained from direct loading of 100 µg of the respective lysates. Under these conditions,
immunoprecipitation with anti-cyclin G2 yielded amounts similar to or
slightly higher than those present in the directly loaded samples (Fig.
3A, bottom; compare lanes
13-15 with lanes 16-18). The
immunosignals for HA-tagged B'
Complementary results were achieved by immunoprecipitation of HA-tagged
B' subunits with anti-HA antibodies and immunoblotting with anti-cyclin
G2 antibodies. Cyclin G2-GFP and G2-V5His fusion proteins were
coimmunoprecipitated with B' subunits from cell lysates of the
B'-cyclin G2 cotransfectants (Fig. 3B, top,
lanes 9, 11, 19, and
20) but not from singly transfected cyclin G2 or B' subunit
expression only controls (Fig. 3B, top,
lanes 6, 7, and 15).
Reprobing this blot with anti-HA antibodies demonstrated again that
relatively more cyclin G2 is associated with B' Colocalization of Cyclin G2 with PP2A B' Subunits in Mammalian
Cells--
To determine whether cyclin G2 and B' subunits are
colocalized in vivo, HEK293 and CHO cells were singly or
doubly transfected with expression constructs for cyclin G2-GFP or
G2-V5His and for HA-tagged B'
Distribution in the cytoplasmic region for cyclin G2 fusion proteins
expressed alone (Fig. 1, B-D) or together with exogenous B'
subunits (Fig. 4) varied between a smooth and a quite punctate pattern.
Similar variations were seen in the staining patterns for HA-tagged
PP2A B' subunits whether expressed alone or in combination with cyclin
G2 (Fig. 4 and data not shown). In CHO cells, a punctate distribution
prevailed for cyclin G2 and B' subunits (Fig. 4), with occasional
smooth distributions (data not shown). Preextraction of HEK293 and CHO
cells with Triton X-100 does not abolish cyclin G2 signals in singly
transfected cells (Fig. 1D and data not shown) or in cells
co-expressing B' subunits (Fig. 4A, lower
panels, B and C), indicating that
cyclin G2 is associated with detergent-resistant subcellular
structures. The same is true for B' subunits coexpressed with GFP
rather than cyclin G2; after preextraction with Triton X-100, GFP
signals were completely gone, indicating that GFP is efficiently
solubilized by the detergent (see Fig. 6F and data not
shown). In contrast to GFP but similar to cyclin G2, PP2A/B' Cyclin G2 Associates with B' but Not A Subunits of
PP2A--
Association of cyclin G2 with B' subunits could reflect the
existence of a cyclin G2 multimeric complex containing the A and C
subunits of PP2A, or it could sequester B' subunits away from a PP2A
core complex. Because B' subunits are thought to target and modulate
the catalytic activity of PP2A/C, it is important to define which
subunits can form complexes with cyclin G2. Since PP2A/A is a
scaffolding protein for B and C subunits, we first tested if endogenous
or overexpressed PP2A/A present in HEK293 lysates interacts with
immobilized cyclin G1- and cyclin G2-GST as found for PP2A/B' subunits.
Several attempts to pull down either endogenous or overexpressed PP2A/A
with immobilized cyclin-GST fusion proteins yielded negative results;
the A subunit did not bind whether ectopically expressed HA tagged B'
subunits were present or not in the lysates (data not shown; see Fig.
6A, upper panels). Next, PP2A/B'
To test whether overexpression of PP2A/A with cyclin G2 could force
their interaction, both proteins were ectopically expressed in HEK293
cells and immunoisolated with anti-cyclin G2 and anti-PP2A antibodies.
Anti-cyclin G2 immunoprecipitates did not contain any detectable PP2A/A
(Fig. 5B, top left panel,
lanes 1, 3, and 5; data not
shown) in contrast to that seen in total cell lysates of both
overexpressed and endogenous PP2A/A (Fig. 5B, top
left, lanes 7-9) and the presence of
abundant amounts of cyclin G2 in the anti-cyclin G2 immunocomplexes
(Fig. 5B, bottom left). Likewise, cyclin G2 was not detectable in anti-PP2A/A immunoprecipitates (Fig.
5B, lower right; data not shown), but
reprobing of these immunoblots with anti-PP2A antibodies demonstrated
the efficient immunoprecipitation of overexpressed and endogenous
PP2A/A (Fig. 5B, top right,
lanes 11, 13, and 15).
Accordingly, PP2A/A appears to be absent from complexes containing
cyclin G2 and vice versa.
Cyclin G2 Exists in a Complex with the C Subunit of PP2A--
To
test whether PP2A/C can interact with cyclin G1 or G2, HEK293 extracts
were incubated with immobilized cyclin G1-GST and G2-GST (Fig.
6A).
Endogenous PP2A/C specifically bound to immobilized cyclin G1-GST and
G2-GST fusion proteins but not control GST. The amount of PP2A/C that
associated with cyclin G1-GST and G2-GST was not significantly altered
by the presence or absence of PP2A/B' subunits (Fig. 6A,
compare left and right bottom
panels). After probing with PP2A/A antibodies, we could not
detect the A subunit in any of the pull-down pellets in contrast to
that in the applied lysates. This observation suggests that endogenous
PP2A/C binding to cyclin G1 or G2 cannot be improved by co-expressing
B' subunits and does not require A subunits. However, it is possible
that unknown endogenous B' subunits are present in saturating amounts and that additional B' subunits cannot increase the binding.
Microcystin is a toxin that specifically and tightly binds to the
catalytic site of both protein phosphatase 1 and PP2A. We used a
microcystin resin to precipitate endogenous PP2A/C from lysates of
cells singly or doubly transfected with expression vectors for cyclin
G2 and HA-tagged B' subunits. Precipitated protein complexes contained
endogenous PP2A/C and cyclin G2 (Fig. 6B). The fraction of
both proteins isolated on microcystin-Sepharose can roughly be
estimated by comparing immunosignals from 50 µg of protein lysate and
the microcystin precipitates from corresponding lysates containing 1 mg
of protein (Fig. 6B, compare lanes 1 to 3 and 4 to 5, etc.). The percentage
of cyclin G2 isolated with endogenous PP2A/C on microcystin-Sepharose
varied between different experiments from ~1% (Fig. 6B,
upper panels, compare lanes
1 and 3) to 5% (Fig. 6B, compare
lane 6 with lane 7 and
lane 10 with lane 11).
Microcystin-Sepharose precipitation isolated about 15-30% of the
total PP2A/C from the lysate regardless of the presence of cyclin G2,
HA-tagged B' subunits singly or in combination (Fig. 6B,
lower panels, compare lanes
1 and 3, etc.). Accordingly, 2-20% of cyclin G2
is associated with endogenous PP2A/C. This amount might be greater if a
substantial amount of cyclin G2 dissociates from the complex during
isolation or is associated with PP2A/C at detergent-resistant sites.
The formation of a complex that contains both endogenous PP2A/C and
cyclin G2 was further confirmed by coimmunoprecipitation of cyclin G2
with anti-PP2A/C antibodies (Fig. 6C). To further examine
whether PP2A/C interacts with cyclin G2 in intact mammalian cells, we
tested whether cyclin G2 colocalized with PP2A/C at distinct sites.
Detection of endogenous PP2A/C in either HEK293 or CHO cells by
immunofluorescence with any of the anti-PP2A/C-specific antibodies
available to us was unsatisfactory. Therefore, we tested whether
HA-tagged PP2A/C can associate with coexpressed cyclin G2-GFP or
G2-V5His in HEK293 cells. HA-PP2A/C coprecipitated with cyclin G2
immunocomplexes isolated from the corresponding cell lysates, and no
signal was detected in immunoprecipitates from control lysates (data
not shown), corroborating that HA-PP2A/C specifically coprecipitated
with cyclin G2. Complementary immunoprecipitation experiments of tagged
PP2A/C with anti-HA antibodies followed by reciprocal blotting with
anti-cyclin G2 antibodies confirmed the association of PP2A/C with
cyclin G2 (data not shown). Immunoblotting with anti-PP2A/C antibodies
indicated that the total amount of PP2A/C was only slightly increased
(~2-fold) in lysates from transfected compared with nontransfected
cell cultures (data not shown).
The majority HA-PP2A/C-transfected HEK293 cells and many transfected
CHO cells exhibited abnormal nuclear morphology (Fig. 6,
D-F, and data not shown), indicating that increased
expression of PP2A/C is not well tolerated, in agreement with previous
reports (46, 55-57). Because expression of HA-PP2A/C was better
tolerated in CHO than HEK293 cells, we used the CHO system to examine
the hypothesized colocalization of cyclin G2 with PP2A/C. When cyclin G2 is co-expressed with HA-PP2A/C, distinct punctate or patchy regions
of colocalization are seen (Fig. 6, D and E).
However, the extent of the overlap varied between different cells
(e.g. compare the two cells coexpressing cyclin G2-GFP and
HA-PP2A/C in the two right panels of
Fig. 6D). The upper right
subpanel in Fig. 6D shows two cells expressing
cyclin G2-GFP (green channel), but only one is
immunoreactive for the HA-tagged PP2A/C (red
channel), showing that the anti-HA staining is specific and
that there is no significant bleed-through between channels. Since
cyclin G2 is associated with Triton X-100-resistant structures when
expressed alone or together with HA-PP2A/B' subunits (see Figs.
1D and 4), we tested whether cyclin G2 is colocalized with
HA-PP2A/C at these detergent-resistant sites (Fig. 6E).
Although a substantial fraction of PP2A/C behaves like a soluble
cytosolic protein, it is also localized in the nucleus, the centrosome,
and other cytoskeletal elements (39, 41, 56, 58, 59). Our
immunocytochemical analysis of CHO cells co-expressing HA-PP2A/C and
GFP, which were either pre-extracted with Triton X-100 buffer (Fig.
6F, left and lower
subpanels) or not (Fig. 6F, upper
right subpanel) is in agreement with these
reports. Whereas GFP was efficiently extracted by the detergent
treatment, a substantial portion of HA-tagged PP2A/C remained and was
localized to the nucleus and punctate perinuclear and cytoplasmic
regions (Fig. 6F). As before (see Figs. 1 and 4), neither
GFP- nor V5His-tagged cyclin G2 was extracted by this pretreatment, but
they were colocalized in puncta with detergent-insoluble HA-PP2A/C to
distinct nuclear, perinuclear, and cytoplasmic regions and along
filamentous structures (Fig. 6E). Of note, in some cells,
the overlap was very strong (e.g. the cell in the
upper right corner of Fig.
6E), and in others it was restricted to a small area
(e.g. the cell in the lower left
corner in the same subpanel). Taken together, the
distribution of cyclin G2 and PP2A/C is clearly, although partially,
overlapping. The degree of overlap appears to be independent of
pre-extraction with Triton X-100 (e.g. Fig. 6, compare
D with E), negating the possibility that
colocalization observed in unextracted cells is simply a reflection of
two abundant overlapping soluble cytoplasmic proteins. These findings
support our hypothesis that these two proteins associate with one
another at distinct subcellular sites in living cells.
Our observations on cyclin G2-PP2A/C associations suggested that this
interaction was not dependent on the presence of exogenous B'
subunits nor the A subunit. These results raised the possibility that the cyclin G2-PP2A/C interaction may be direct. To further evaluate this point, PP2A complexes were isolated from
HA-PP2A/C-transfected cells with anti-HA antibodies. Immunocomplexes
were dissociated with SDS at 60 °C, and SDS was neutralized with an
excess of Triton X-100. Aliquots of these samples were used for
pull-down experiments with cyclin G1-GST, G2-GST, and GST alone bound
to glutathione-Sepharose. HA-tagged PP2A/C associated with cyclin G1-
and G2-GST but not GST alone (Fig.
7A, lower
right panel), indicating that the C subunits can
directly interact with these two cyclins. Again, PP2A/A was not
detectable in the PP2A/C cyclin G2-interacting complexes (Fig.
7A, upper panel).
This SDS dissociation protocol has been used for other protein
complexes without detecting reassociation (47, 48). Furthermore, the
interaction between immunoisolated HA-PP2A/C and immobilized cyclin G1-
and G2-GST did not require the presence of co-expressed HA-tagged B'
subunits. It remains possible that an unknown endogenous B' subunit
acts as a bridge between immobilized cyclin G1- or G2-GST and soluble
HA-PP2A/C. To rule out the requirement of B' subunits for cyclin
G2-PP2A/C association, we expressed PP2A/C as a GST fusion protein with
and without an HA tag in E. coli and immobilized them on
glutathione-Sepharose resins (Fig. 7, B and C).
These, along with control GST resins, were used as targets for
interaction with bacterially expressed polyhistidine-tagged cyclin G2
and nonrelevant control proteins in pull-down assays (Fig.
7D). Bacterially expressed cyclin G2 specifically binds to
PP2A/C-GST fusion proteins (Fig. 7D, lanes
5 and 6) but not control GST (lane
7) in the absence of any other PP2A subunits. These data
demonstrate that cyclin G2 can directly interact with PP2A/C. Since
microcystin-Sepharose coprecipitates cyclin G2 with PP2A/C (Fig.
6B), this interaction is probably independent of the PP2A/C
catalytic site.
PP2A/C Phosphatase Activity Associates with Endogenous and Ectopic
Cyclin G2 Complexes--
We tested whether PP2A/C associated with
cyclin G2 is catalytically active. Anti-cyclin G2 and control IgG
immunocomplexes were isolated from lysates of CHO and HEK293 cultures
transfected with either cyclin G2 or control GFP. Immunocomplexes were
incubated with a PP2A/C phosphopeptide substrate, and the amount of
released phosphate was measured. A substantial amount of PP2A activity (as determined by its sensitivity to both 1 µM
microcystin and okadaic acid (20 and 2 nM)) coprecipitated
with cyclin G2 but not with control IgG (Fig.
8, A and B). In
parallel, the presence of PP2A/C in cyclin G2 immunocomplexes isolated
from cyclin G2 transfectants (but not in control IgG or anti-cyclin G2
mock immunoprecipitations from GFP controls) was confirmed by
immunoblotting (Fig. 8, A and B). Measurement of
phosphatase activity associated with PP2A/C immunocomplexes containing
similar amounts of PP2A/C isolated from cyclin G2-expressing and
nonexpressing cultures determined that they contained nearly equal
levels of activity (data not shown).
Since PP2A/A is the predominant cellular scaffolding subunit for PP2A/C
but is not present in cyclin G2-PP2A/C complexes, we compared the
relative activity of PP2A/C associated with endogenous PP2A/A with that
associated with cyclin G2. PP2A holoenzymes complexed with endogenous
PP2A/A were immunoisolated with 6F9 rat anti-PP2A/A antibodies from
lysates of HEK293 cultures expressing PP2A/B'
To test whether endogenous cyclin G2 is associated with PP2A/C, we
immunoisolated cyclin G2 from rat brain cerebellum, a tissue where
cyclin G2 is abundant. The presence of PP2A/C protein and activity was
determined by immunoblotting and the phosphatase assay as described
above. Not only was endogenous PP2A/C associated with endogenous cyclin
G2, this complex dephosphorylated the phosphopeptide substrate, and the
activity could be inhibited by more than 50% in the presence of 2 nM okadaic acid (Fig. 8C). Thus, a population of
functionally active endogenous PP2A can be found associated with
endogenous cyclin G2.
The Carboxyl-terminal Region of Cyclin G2 beyond the Cyclin Box
Motif Is Necessary and Sufficient for Association with PP2A--
All
cyclins share a 110-amino acid homologous region called the cyclin box
that is essential for CDK binding and activation (60, 61). We wanted to
know whether the cyclin box region of cyclin G2 containing amino acid
sequences required for complex formation with PP2A subunits can mediate
the interaction with PP2A. Amino- and carboxyl-terminally deleted forms
of cyclin G2 were expressed as GFP chimeras together with HA-tagged
subunits of PP2A in HEK293 cells. Anti-HA immunoblots of anti-cyclin G2 immunoprecipitates from HEK lysates cotransfected with HA-B' Sequences Carboxyl-terminal to the Cyclin G2 Cyclin Box Are
Necessary and Sufficient to Induce Cell Cycle Arrest--
Since
moderate to high levels of ectopic cyclin G2 protein can be
antagonistic to cell cycle progression and amino acids 142-241 of
cyclin G2 are important for interaction with PP2A B' and catalytic subunits, we wanted to test whether this region of cyclin G2 can elicit
the same negative effects on the cell cycle as full-length cyclin G2 or
if a region not important for PP2A interaction mediates these
inhibitory effects. We expressed various NH2- or
COOH-terminal regions of cyclin G2 as GFP chimeras and full-length
cyclin G2 in a GFP chimeric or nonchimeric form as well as control GFP
in HEK 293 and CHO cells and subjected the individual transfected cultures to flow cytometric cell cycle analysis as described above. Comparison of the cell cycle profile for nonexpressing (no GFP signal)
with that of expressing cell populations (GFP-positive) indicated that
the regions found to interact with PP2A (residues 142-344 and 1-241)
have cell cycle-inhibitory effects (Fig. 9, table) similar
to full-length cyclin G2; those truncated cyclin G2 chimeras that do
not interact well with PP2A (CycG2 55-140 and 1-140) have no or
little effect on cell cycle progression (Fig. 9, table).
Like full-length cyclin G2 (expressed either as a GFP chimera or as a
nonfused independently translated form), expression of the cyclin G2GFP
chimeras containing residues 142-241 resulted in a G1
phase cell cycle arrest. These findings were further confirmed by
BrdUrd incorporation; cells expressing the NH2-terminal
1-140 residues of cyclin G2 had normal levels of BrdUrd incorporation,
but cells expressing residues 142-344 of cyclin G2 exhibited low
levels of DNA synthesis (data not shown). These results suggest that
the negative effects of ectopic cyclin G2 expression on cell cycle
progression are linked to formation of a cyclin G2-PP2A complex.
Our attempts to obtain, in repeated trials, HEK293 or CHO cell
clones stably expressing cyclin G2 suggested that unregulated cyclin G2
expression is deleterious. Indeed, in transiently transfected HEK293,
CHO, and SAOS-2 cultures, we found aberrant nuclear morphology in
30-60% of the cells expressing high levels of cyclin G2 and the
induction of G1/S phase cell cycle arrest in the cyclin G2 transfectant population. The preponderance of aberrant nuclei in cyclin
G2 transfectants within 28-48 h post-transfection did not contain
hypercondensed chromatin, were TUNEL-negative, and were surrounded by
intact lamin B-positive nuclear envelopes. These observations suggest
that the initial impact of unlicensed cyclin G2 expression was a
dysregulation of a cellular division process leading to an aberrant
mitosis/cytokinesis followed by cell cycle arrest rather than a direct
induction of apoptosis. Flow cytometry of sorted populations indicated
an accumulation of cyclin G2 expressors in the G1 phase of
the cell cycle. Cell populations expressing moderate to high levels of
cyclin G2 exhibited a much reduced level of CDK2 kinase activity and
DNA synthesis but a high level of CDK4 activity, consistent with a
block between the mid-G1 and G1/S phase
boundary. Because typical cyclins interact with CDKs, this phenotype
might have resulted from cyclin G2 dysregulation of CDK partners. We
did not detect any kinase activity in cyclin G2 immunoprecipitates
under standard phosphorylation conditions toward histone H1, a classic
in vitro CDK substrate, although such activity was
associated with positive control immunoprecipitates of CDK2 and cyclin
B1 in our hands.4
Furthermore, probing immunoblots of cyclin G2 immunoprecipitates yielded negative results for various CDKs including CDK1, -2, and
-4.4 It is possible that histone H1 is a poor substrate for
a putative cyclin G2-associated CDK (as is the case for cyclin D-CDK4
and -CDK6 complexes (62)) or that cyclin G2 activation of a CDK occurs
only in response to specific signals. Although these negative results
cannot rule out that cyclin G2 is associated with a kinase, they
prompted us to investigated alternative interaction partners of cyclin G2.
Cyclin G1 interacts with the murine PP2A B' subunits termed B' The cyclin G2-PP2A-mediated induction of G1 phase cell
cycle arrest with active CDK4 and inactive CDK2 could be due to
dephosphorylation of either Rb or its relatives p107 and p130 (63).
Their dephosphorylation would result in sequestration of E2F
transcription factors, thereby preventing activation of CDK2 and
progression of the cell cycle. Although PP2A has been implicated in the
phosphorylation/dephosphorylation of Rb-related pocket proteins, the
form of PP2A found to regulate p107 contains the B" subunit PR59 and
not a B' subunit (25). Furthermore, protein phosphatase 1 is believed
to be the primary Rb regulatory phosphatase (64, 65). Alternatively,
the cyclin G2-PP2A complex could function to directly dephosphorylate
CDK2 at Thr160 and thus prevent its full activation, but we
were unable to detect endogenous CDK2 in cyclin G2 immunocomplexes. It
is also possible that the lack of CDK2 activation is more indirect and
the result of cyclin G2-PP2A inhibitory dephosphorylation activity
toward the CDK2-activating phosphatase CDC25 (26, 66).
The presence of aberrant nuclei seen upon overexpression of cyclin G2
suggests a dysregulation of a late mitotic or cytokinetic process. This
dysregulation constitutes an alternative mechanism that could
contribute to the observed G1 phase arrest. Aberrant cytokinesis has been shown to induce G1 phase cell cycle
arrest (67, 68). Functional centrosomes are needed not only for the fidelity of cytokinesis but also for cell cycle progression through G1 into S phase (69-71). A portion of PP2A/C is localized
to microtubules, intermediate filaments, and centrosomes (16, 39, 41,
59, 72). Mitotic and cytokinetic defects have been linked to
dysregulated or mutated PP2A subunits in yeast, Drosophila,
and mammalian cells (57, 73-77). Mutation of PP2A/C results in
uncoupling of the nuclear and centrosomal cycles in
Drosophila (56), and inhibition of serine/threonine
phosphatases in CHO cells induces abnormal centrosome duplication,
resulting in aberrant mitosis (78). We determined that cyclin G2 and
PP2A/C are colocalized in Triton X-100-preextracted cells at dense
areas within nuclei and between fragmented nuclei and in some cases at
distinct perinuclear dotlike structures suggestive of centrosomes. The
effects of ectopic cyclin G2 expression on nuclear structure and the
cell cycle suggest, therefore, a role for the cyclin G2-PP2A/C complex
in regulation of cytoskeletal processes central to cellular division.
Mutations or deletions in certain PP2A regulatory B/B' subunits in
yeast and flies or displacement of B subunits by viral transforming
antigens result in alterations in cell growth and cell division (22,
37, 74, 76, 79). Mutation of the single Saccharomyces
cerevisiae B' subunit, Rts1p, and of the two
Schizosaccharomyces pombe B' subunits Par1 and Par2
leads to cytokinetic defects (79, 80). Sequestration of B' subunits by
cyclin G2 would probably elicit effects similar to those in B' subunit
deletion mutants. Accordingly, overexpression of cyclin G2 complexed
with B' subunits could induce subtle changes in the subcellular
distribution of PP2A/B'-C complexes, leading to an altered
regulation of cellular division resulting in aberrant nuclei and
ultimately a G1/S phase arrest. Ectopic co-expression of B'
or C subunits with cyclin G2 does not appear to dramatically influence
the distribution of the latter in HEK293 or CHO cells. However, it is
possible that a substantial amount of cyclin G2 is recruited to certain
subcellular structures via an interaction with other proteins, possibly
yet to be identified endogenous B' subunits, occluding relocalization
upon coexpression with PP2A/B' Carboxylmethylation of PP2A/C is a cell cycle-regulated and reversible
modification enhancing its activity and fostering the binding of the
B We determined that both ectopic and endogenous cyclin G2-C subunit
complexes exhibit an okadaic acid-sensitive (<2 nM)
phosphatase activity. Although a relatively small fraction of the total
PP2A/C pool coprecipitated with cyclin G2 from transfected cell lines and rat brain homogenates, this does not argue against physiological significance. PP2A is an abundant essential phosphatase fulfilling a
multitude of cellular functions forming different complexes with
various subunits and target proteins. Several specific protein complexes containing only a small fraction of total PP2A/C are predicted or known to have important cellular functions (16, 17, 19,
84). Analogous to the CaMKIV-PP2A complex (19), the cyclin G2-PP2A/C
complex can be isolated with microcystin-Sepharose. Because microcystin
binds to the catalytic site of PP2A/C, this interaction did not require
phosphatase activity or access to the catalytic site itself and
suggests that PP2A/C-cyclin G2 association is not simply an interaction
between the catalytic site of the C subunit and a substrate. In
parallel experiments, neither endogenous nor overexpressed PP2A/A was
found in cyclin G2-PP2A/B'-C subunit immunocomplexes, and PP2A/A did
not interact with cyclin G2 in pull-down assays. These results offer
the prospect that cyclin G2 and G1 can act as scaffolding or adapter
proteins for PP2A/B' and C subunits. Our analysis of phosphatase
activity associated with PP2A/A-anchored holoenzymes compared with
cyclin G2-PP2A/C complexes suggests that high levels of cyclin G2 can
compete with PP2A/A for a portion of PP2A/C. It is possible that B'
subunits stabilize PP2A/C interactions with cyclin G2, although the
amount of PP2A/C associated with cyclin G2 in lysates did not depend on
the presence of exogenous HA-tagged B' subunits in HEK293 cells. Nevertheless, an unidentified B' subunit that associates with cyclin G2
as well as PP2A/C may be present in these cells and thereby target the
heterotrimeric cyclin G2-PP2A/B'-C complex to distinct subcellular sites.
The expression level of PP2A/C is tightly regulated by translational
control and post-translational modifications; significant alterations
in the level of available PP2A/C is difficult to achieve without
deleterious effects on cell growth and survival (46, 55-57). During
Fas-mediated apoptosis the PP2A/A subunit is cleaved by
caspase-3-related proteases, and the activity of PP2A/C is increased
(85). PP2A activity is also increased during the execution phase of
apoptosis in neuronal cell lines (86) and by the sphingolipid ceramide
(87-89), a putative second messenger in various apoptotic responses,
including those of immature B lymphocytes (90-92). Cyclin G2 is
up-regulated in immature B cell lines during growth arrest and
apoptosis, concurrent with increases in ceramide and caspase-3 family
member activation (9, 91, 93). We have found PP2A/C in cyclin G2
immunocomplexes isolated from stimulated B cell lines expressing high
levels of cyclin G2.4 It is plausible that cyclin G2 could
replace caspase targeted PP2A/A and form a ternary complex with free B'
and C subunits. The cyclin G2-PP2A complex might possess differentially
modulated activity important for the final execution steps of apoptosis.
Cyclin G2 is highly expressed in the brain (8, 9), where it forms
catalytically active complexes with PP2A C subunits (Fig. 8). Although
we do not know the type of B regulatory subunit present in cyclin G2
complexes in the cerebellum, it is probably a B' subunit.
Differentiation signals up-regulate the expression of brain-specific B'
subunit isoforms in neuroblastoma cells (38), which are thought to
direct PP2A to particular compartments in response to signal
transduction during brain development. Our finding that cyclin G2 and
PP2A/C can directly interact and form enzymatically active complexes
suggests that cyclin G2 is involved in modulating either the
subcellular localization or specificity of the PP2A/C complex, perhaps
in conjunction with specific B' subunits. The formation of this cyclin
G2 complex is inhibitory for cell cycle progression and may be
important to promote sustained exit from the cell cycle in response to
inhibitory signals or cellular differentiation programs.
We thank Drs. Johannes W. Hell and Dawn E. Quelle for advice, discussion, and a critical reading of the
manuscript. We express our gratitude to Dr. Brian E. Wadzinski
(Vanderbilt University, Nashville, TN) for the sheep and rabbit
anti-PP2A/C antibodies, for the pcDNA3 expression vector encoding
HA-tagged (and untagged) PP2A/C, and for advice and discussion and Dr.
Gernot Walter (University of California, San Diego, CA) for the
generous gift of the monoclonal antibodies 6G3 and 6F9 against PP2A/A.
Kathleen Schell of the University of Wisconsin Comprehensive Cancer
Center Flow Cytometry Facility is thanked for expert technical
assistance with cell sorting and FACS analysis as well as excellent
advice and consultation. We express our appreciation to Robert
Creighton for assistance in antibody production and testing.
*
This work was supported by National Institutes of Health
Grant R01GM56900 and American Heart Association Grant 9806386X (to M. C. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, April 15, 2002, DOI 10.1074/jbc.M111693200
2
D. A. Bennin, A. S. Arachchige Don, H. Rosenbaum, and M. C. Horne, unpublished results.
3
M. C. Horne, unpublished data.
4
D. A. Bennin and M. C. Horne,
unpublished data.
The abbreviations used are:
CDK, cyclin-dependent kinase;
FITC, fluorescein
isothiocyanate;
GFP, green fluorescent protein;
GST, glutathione
S-transferase;
HRP, horseradish peroxidase;
LRSC, lissamine
rhodamine sulfonyl chloride;
PBS, phosphate-buffered saline;
TBS, Tris-buffered saline;
PP2A, protein phosphatase 2A;
HA, hemagglutinin;
PIPES, 1,4-piperazinediethanesulfonic acid;
FACS, fluorescence-activated cell sorting;
PI, propidium iodide;
BrdUrd, bromodeoxyuridine;
CHO, Chinese hamster ovary;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling.
Cyclin G2 Associates with Protein Phosphatase 2A Catalytic
and Regulatory B' Subunits in Active Complexes and Induces Nuclear
Aberrations and a G1/S Phase Cell Cycle Arrest*
,§,,,,,§
Department of Pharmacology, University of
Wisconsin, Madison, Wisconsin 53706-1532, the § Department
of Pharmacology, University of Iowa, Iowa City, Iowa 52242-1109, and
the ¶ Department of Biochemistry and Molecular Biology, Indiana
University, Indianapolis, Indiana 46202-5122
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
light chain was
from Zymed Laboratories Inc. (South San Francisco,
CA). All other reagents were of standard quality from established suppliers.
-mercaptoethanol (His), 10 mM EDTA (GST proteins only), and 1.5% sarkosyl were added.
Insoluble material was removed by centrifugation (30,000 rpm, 60 min,
4 °C, 45Ti rotor), and supernatants were frozen and stored at
80 °C. For affinity purification, bacterial lysates were thawed
and diluted 1:5 in TBS containing 2-3% Triton X-100 to neutralize sarkosyl. Lysates were incubated with 1 ml of glutathione-Sepharose (GST proteins) or Ni2+-nitrilotriacetic acid resin (Qiagen;
His) at 4 °C for 2 h. The resins were washed three times with
TBS containing 0.05% Tween 20 and once with TBS only. The purity of
all immobilized proteins was confirmed by SDS-PAGE and Coomassie
staining, revealing a single major band at the predicted molecular
mass. The identity of each fusion protein was further confirmed by
immunoblot analysis with anti-GST and anti-T7 antibodies (47), with
antibodies produced against multiple antigen peptides derived from
NH2-terminal sequences of cyclin G1 and G2, respectively
(see below),3 and with
anti-PP2A/C antibodies. For elution of cyclin G1- and G2-TrcHis,
200-500 mM imidazole was added to the washing buffer.
2, B'3, or B'4 or pCMVneoHA B'
(31)), for NH2-terminally HA-tagged PP2A/C
subunit (HA-PP2A/C in pcDNA3+ kindly provided by Dr. Brian E. Wadzinski, Vanderbilt University, Nashville, TN (46)) or rabbit
skeletal muscle PP2A/A in pcDNA3 or a combination of these
constructs. An equal molar amount of pAdVantage (Promega) was included
in each transfection mix to enhance transfection and translation efficiency (52). Cells were harvested 24-36 h post-transfection.
-glycerol phosphate, 10 mM EGTA, 20 mM NaPPi, and 2 µM microcystin), and the lysates
were cleared of insoluble material by centrifugation at 21,000 × g for 10 min. Immunoprecipitations of CDK2 were performed in duplicate
from cleared supernatants containing equivalent amounts (80 or 100 µg
each) of protein using 10 µg of rabbit anti-CDK2 antibodies (sc-54;
Santa Cruz Biotechnology). The anti-CDK2 and control immunocomplexes were washed three times with buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, and 5 mM
MnCl2), incubated for 30 min at 37 °C in 40 µl of
kinase buffer (50 mM Hepes, pH 7.4, 10 mM
MgCl2, and 5 mM MnCl2, 1 mM dithiothreitol, 10 µM ATP, 2 µg of
histone H1 (Calbiochem) supplemented with 10 µCi of
[-32P]ATP). Reactions were analyzed by SDS-PAGE and
autoradiography. In parallel, duplicate CDK2 and control
immunocomplexes and total lysate (80 µg of protein) were
immunoblotted with anti-CDK2.
-mercaptoethanol, 0.066% bovine
serum albumin) for 2 h. 45 µl of the reaction eluate was
collected after centrifugation and added to a 96-well microtiter plate
followed by the addition of an equal volume of the molybdate/malachite green dye. After 15 min at room temperature, absorbance was measured at
600 nm. The amount of phosphate released in the immunocomplexes was
calculated from a curve of phosphate standards run in parallel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of anti-cyclin G2 antibodies
and the effect of cyclin G2 overexpression. HEK293 or CHO cells
were transfected with pcDNA3 expression constructs encoding
either G2-GFP, G2-V5His, or GFP alone. A, for
immunoblotting, 50 µg of protein from HEK293 lysates cleared by
centrifugation (lanes 1-3) and 5-10 ng of
affinity-purified GST fusion proteins (lanes
4-6) were loaded as indicated. Blots were probed with
affinity-purified anti-cyclin G2 antibody, followed by HRP-conjugated
protein A. The migrations of the immunoreactive fusion proteins present
in each lane are indicated by the arrows, and the apparent
molecular masses of the protein standards are depicted at the
left side of the blot. B-D,
immunofluorescence confocal microscopy of cyclin G2-expressing HEK293
(B and C) or CHO (D) cell cultures.
Cultures expressing cyclin G2-V5His (B (left
side of panels) and D) or
cyclin G2-GFP (B (right sides) and
C) were fixed either before permeabilization with Triton
X-100 (B and C) or after preextraction with
Triton X-100 (D) and stained with affinity-purified
anti-cyclin G2 antibody (red), followed by LRSC-conjugated
donkey anti-rabbit IgG (B (middle) and
D (left)), with anti-V5His, followed by
FITC-conjugated donkey anti-mouse IgG (B (left
side of left panel) and D
(middle)), with anti-lamin B antibodies in conjunction with
LRSC-conjugated donkey anti-goat IgG (C (red in
left panel); anti-lamin B immunosignals are also
separately shown in the middle panel in
black and white for better contrast),
and with TOTO-3 for DNA detection in transfected and untransfected
cells (pseudocolored blue in B (right)
and C (left) and shown in black
and white in D (right)).
The percentages of cells with abnormal nuclei upon cyclin G2 expression
in experiments represented by the images were 50%
(B, left) and 64% (B,
right), 55% (C), and 54% (D).
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Fig. 2.
Flow cytometry and cell cycle analysis of
cyclin G2-expressing cells. A, analysis of DNA content
and GFP signal intensity by flow cytometry of intact (propidium
iodide-negative) live CHO cells transfected with either cyclin G2GFP or
control GFP expression plasmids, stained with the cell-permeant DNA dye
Hoechst 33342, 21 h post-transfection. The amounts of DNA
(Hoechst) relative to the expressed GFP signal in a total population of
transfected cells are shown as dot plots (upper
panels) and DNA profiles of the expressing versus
nonexpressing gated populations in the labeled histograms
below each corresponding dot plot. We determined the
percentage of cells in the G1, S, and G2 + M
phases (given at the right for each DNA histogram with the
FlowJo Watson Pragmatic cell cycle analysis program; similar results
were obtained applying the Dean-Jett-Fox cell cycle modeling
algorithm). B, bivariate flow cytometric analysis of the DNA
content (propidium iodide) versus GFP signal and new DNA
synthesis (BrdUrd incorporation detected with an anti-BrdUrd primary
and Alexa 660 fluorophore-labeled secondary antibody) of CHO cells
transfected with cyclin G2GFP DNA (27 h) in dot plot format. The
left panel shows the DNA profile of the total
transfected culture and the population gates set for analysis of BrdUrd
incorporation shown in corresponding labeled dot plots in the
right two panels. The percentage of
cells incorporating the BrdUrd label into DNA during the 15-min pulse
of each population is shown to the upper right of
the correspondingly shaded gate (identical for both populations), and
the calculated cell cycle distribution derived from the total DNA dot
plot is shown on the side. C, CDK2 and CDK4
kinase activity of FACS-sorted live populations of CHO cells expressing
or not expressing either cyclin G2GFP or GFP. The upper and
middle panels on the left and
lowest panel on the right are
autoradiograms of 32P-labeled histone H1 phosphorylated by
CDK2 isolated by immunoprecipitation from identical amounts (80 µg)
of lysates prepared from the nonexpressors (no), expressors
(high), and total population of cells, as indicated. No
kinase activity was detectable after immunoprecipitation with control
nonimmune IgG (NRS IgG; middle panel
on the left). Bottom panel on
left, anti-CDK2 immunoblot of lysate (right) and
CDK2 immunoprecipitated in parallel from identical amounts of the same
extract used in the kinase assay shown in the middle
panel. Quantification of the above signals indicated an
approximate 7-13-fold reduction of CDK2 kinase activity in cyclin
G2-expressing cells. The top panel on the
right shows the specificity of CDK4 immune complexes
compared with control IgG for RbGST phosphorylation (autoradiogram of
32P-labeled Rb-GST). The middle two
panels on the right are the autoradiogram of the
CDK4 activity toward Rb-GST in anti-CDK4-immunoprecipitates from sorted
and total population lysates (indicated at the top of the
right panels; 200 µg of protein) and the
respective CDK4 immunoblot signals. In parallel, a determination of
histone H1 activity in anti-CDK2 immunoprecipitates (lower
right) was obtained from the same lysates (100 µg of
protein) of the respective sorter populations used for the CDK4
assay.
-32P]ATP followed by SDS-PAGE and
autoradiography to test for CDK2 activity (Fig. 2C,
upper panel). CDK2 activity immunoprecipitated from cyclin G2GFP-expressing CHO cells was significantly reduced in
comparison with the corresponding population of control GFP expressors
and nonexpressing cell populations of each transfection (Fig.
2C, upper left). Similar results were
obtained in HEK293 cells (data not shown). Next, the
CDK2-dependent histone H1 kinase activity of CHO cell
cyclin G2GFP-expressing and nonexpressing populations was compared with
the amount of CDK2 immunoprecipitated and present in each lysate (Fig.
2C, lower left panels). No
kinase activity was precipitated by preimmune antisera
(NRS), and again the cyclin G2GFP-positive population
contained a dramatically reduced CDK2 activity. Quantification of the
kinase activity relative to the amount of CDK2 immunoprecipitated or
present in total lysate indicated about a 10-fold reduction in
CDK2-specific kinase activity in the cyclin G2-expressing population
compared with the nonexpressors (Fig. 2C, lower
left two panels). To determine whether
the cell cycle block in cyclin G2-expressing cells was in early
G1 phase or at the G1/S phase boundary, a
similar analysis of CDK4 activity was performed using Rb-GST as a
kinase substrate. CDK4 activity is critical for entry into and
transition through the early G1 phase. Interestingly,
cyclin G2 expressors contained increased levels of CDK4 activity
relative to that in nonexpressors and the total population (Fig.
2C, right panels), probably due to the
accumulation of cells in the early G1 phase. These data
indicate that cyclin G2-expressing CHO and HEK293 cells can enter and
progress through early G1 phase but cannot effectively move
beyond the G1/S phase of the cell cycle.
3 and B' Subunits in
Living Cells--
We were unable to find any typical CDK activity or a
cell cycle-related CDK associated with cyclin G2. Because cyclin G1
associates with B' regulatory subunits of PP2A, an enzyme that also
plays a critical role in cell cycle progression, we tested for cyclin G2-PP2A interactions. PP2A B' subunits are encoded by six different genes that give rise to at least nine distinct proteins, including splice variants. Varying nomenclatures categorizing these proteins have
been instituted (31, 34, 35, 38, 42). Csortos et al. (31)
cloned and described four different rabbit B' subunits (B'
-
),
including B' and B' subunits. Okamoto et al.
determined that two murine B' subunits interact with cyclin G1, which
they termed B' and B' (44). These are the respective murine
homologs of the rabbit B' and B' subunits (31).
and B' subunits (nomenclature established
in Ref. 31). Initially purified and immobilized GST, cyclin G2-GST,
cyclin G1-GST, and polyhistidine-tagged cyclin G1 and cyclin G2 fusion
proteins were tested in vitro for interactions with
HA-tagged B' or B' splice variant B'2, B'3, or B'4 (31) present in transfected cell lysates. All of the HA-tagged subunits bound to cyclin G2 as well as cyclin G1-GST and polyhistidine-tagged fusion proteins but not to control GST (data not shown). These results
indicated that in vitro, cyclin G2 interacts with splice variants of PP2A/B' and PP2A/B' and that cyclin G1 binds not only
B' and splice forms of B' but also B' subunits.
3 and B', respectively, were present in cyclin G2
immunocomplexes isolated from the corresponding cyclin G2-PP2A/B'
cotransfectants (Fig. 3A,
top, lanes 5, 6,
8, and 9) but not in those from control GFP-PP2A/B' cotransfectants (Fig. 3A, top,
lanes 2 and 3) or singly transfected
controls (Fig. 3A, top, lanes
1, 4, and 7). Immunoprecipitates isolated with GFP-specific antibodies from control cultures transfected with an expression vector for GFP with or without vectors for the
HA-tagged B' subunits did not contain any HA-immunoreactive proteins
(Fig. 3A, top, lanes
10-12). This experiment also suggests that B'
coprecipitated with cyclin G2 more efficiently than B'3; significantly more PP2A/B' was present in cyclin G2 immunocomplexes isolated from G2-GFP-transfectant lysates (Fig. 3A,
top, lanes 5 and 6) despite
the presence of similar amounts of GFP-tagged G2 in these samples (Fig.
3A, bottom, lanes 5 and
6). Although much smaller amounts of cyclin G2
immunoprecipitated from lysates of the cyclin G2-V5His plus B'
cotransfectants (relative to the B'3 cotransfectants), similar
amounts of these two HA-tagged B' subunits copurified with the two
cyclin G2 immunocomplexes (Fig. 3A, bottom,
lanes 8 and 9).
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Fig. 3.
Co-immunoprecipitation of cyclin G2 with PP2A
B' subunits. HEK293 cells were co-transfected with DNA constructs
encoding GFP, G2-GFP, or G2-V5His and either an irrelevant protein
(Cont.) or various HA-tagged PP2A B' subunits as indicated
at the top of each panel. A, cleared
cell lysates containing 400 µg (lanes 1-12),
500 µg (lanes 13-15), or 1 mg of total protein
(lanes 19-21) were used for immunoprecipitations
with anti-G2 or anti-GFP antibodies as depicted at the
bottom (IP). Lysates containing 200 µg
(lanes 16-18) or 50 µg of protein
(lanes 22 and 23) were also directly
loaded. Immunoblots were incubated with anti-HA antibodies
(top panels) followed by either HRP-conjugated
goat anti-mouse IgG (lanes 1-18) or rat
anti-mouse Ig light chain (lanes 19-23). The
latter was used to probe for PP2A/B'4, which migrates close to the
IgG heavy chain, to avoid detection of the heavy chain of the
immunoprecipitating mouse anti-HA antibody (as observed in some
exposures of blots probed with the goat anti-mouse IgG; e.g.
the band at 50 kDa in lanes 1-12). Blots were
stripped with SDS (53) and reprobed with anti-cyclin G2 antibodies,
followed by HRP-labeled protein A (bottom
panels). B, cleared cell lysates containing 1 mg
(lanes 1-4 and 18-20) or 800 µg of
total protein (lanes 5-11) were used for
immunoprecipitations with anti-G2, anti-HA, or control antibodies as
given at the bottom (IP). Lysates containing 100 µg (lanes 12-14) or 50 µg of protein
(lanes 15-17) were also directly loaded. Blots
were incubated with anti-cyclin G2 antibodies, followed by HRP-labeled
protein A (top panels). Blots were stripped with
SDS (53) and reprobed with anti-HA (bottom
panels) followed by either HRP-conjugated goat anti-mouse
IgG (lanes 1-14) or rat anti-mouse Ig light
chain (lanes 15-20) to avoid detection of the
heavy chain of the immunoprecipitating antibody when probing for
PP2A/B'4, which migrates close to the IgG heavy chain. Positions of
marker proteins are depicted at the left or right
of each blot (in kDa).
3 and B' subunits coprecipitated
with cyclin G2 are roughly 10-20% and above 50%, respectively, of
those in the corresponding whole lysates (Fig. 3A,
top; compare lanes 14 and
15 with lanes 17 and 18).
Collectively, these results suggest that 10-20% of HA-tagged
PP2A/B'3 and 50% or more of HA-tagged PP2A/B' in these lysates
are associated with cyclin G2. HA-tagged PP2A/B'4
coimmunoprecipitated with cyclin G2-V5His and G2-GFP, and the extent of
these interactions is similar to that between cyclin G2 and PP2A/B'3
(Fig. 3A, lanes 19-23).
than with B'3 in
transfected cell extracts; the cyclin G2 signal is several times
stronger after immunoprecipitation from B' than from B'3
cotransfectants (Fig. 3B, top, lanes
9 and 11), although the anti-HA signal is
comparatively less in the former than in the latter sample (Fig.
3B, bottom, lanes 9 and
11).
, B'4 or B'3 (Fig.
4). Similar to cyclin G2 (see Fig. 1,
B-D), immunoreactivity for HA-tagged B' subunits is present throughout the cytoplasmic region and often in the nucleus, whether expressed with (Fig. 4A, upper panels)
or without (Fig. 4, B and C) cyclin G2. Both
B'3 and B' contain classic bipartite nuclear localization signals
in their COOH termini, and the human B' homolog has been shown to be
both nuclear and cytoplasmic (31, 38, 40, 42). Although B'4 lacks
this nuclear targeting sequence, it still efficiently localizes to the
nucleus (31, 38). Clear examples of nuclear colocalization are seen in
the cyclin G2-PP2A/B'- and B'3-cotransfected CHO cells shown in Fig 4, D-F. Although nuclear colocalization was more
pronounced in CHO cells, it was also observed in several HEK293
cotransfectants (data not shown). B' and B' partially rescue the
growth defect of the mutant Rts1p, a B' subunit in yeast (43). Rts1p is
a cytoplasmic protein, whereas B' is both nuclear and cytoplasmic. B' complementation of the Rts1p mutant results in abnormal
morphology (43). Of note, in some cases (Fig. 4 and data not shown)
cells that overexpress PP2A/B' or - contained multilobed or
fragmented nuclei similar to cyclin G2-GFP- or G2-V5His-expressing
cells (Fig. 1, B-D). The observations that overexpression
of either cyclin G2 or B' subunits often results in comparable
nuclear aberrations suggest that they are involved in coordinating
similar processes.
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Fig. 4.
Colocalization of PP2A B' subunits with
cyclin G2 by immunofluorescence confocal microscopy. CHO cells
were transfected with plasmids for expression of HA-tagged
(A, red) B' 3 or B' (B and
C, red) and cyclin G2-GFP (A and
B, green) or G2-V5His (C,
green). After 24-29 h, cells were fixed either before
permeabilization with Triton X-100 (A, upper
panel) or after preextraction with Triton X-100
(A (lower panel), B, and
C). Cultures were stained with anti-HA antibodies followed
by LRSC-conjugated donkey anti-mouse IgG to visualize HA-tagged
PP2A/B'4 and B' and with TOTO-3 for labeling of DNA
(blue channel in right
panels). Those cultures co-expressing V5His-tagged cyclin G2
were also labeled with anti-cyclin G2 antibodies, followed by
FITC-conjugated donkey anti-rabbit IgG (C).
immunoreactivity was still present after preextraction (data not shown). The same was true for PP2A/B'3 expressed with
(e.g. Fig. 4A, lower panel)
or without cyclin G2 (data not shown). G2-GFP- or G2-V5His-positive
dots (Fig. 4) often, though not always, matched those for B'3 (Fig.
4A) or (Fig. 4, B and C), whether
cells were preextracted with Triton X-100 (Fig. 4, A,
lower panel, B, and C) or
not (Fig. 4A, upper panel). In
general, preextraction did not change the staining pattern for either
cyclin G2 or B' subunits (e.g. compare the unextracted cell
in Fig. 4A, upper panel, with the
preextracted cells in Fig. 4B). Untransfected cells
(visualized with the TOTO-3 DNA stain; blue
channel in right panels in Fig. 4) did
not show any immunofluorescence for anti-HA or anti-cyclin G2
antibodies, confirming the antibody specificities. Collectively, our
data show that both cyclin G2 and B' subunits are to a significant
degree colocalized with each other before and after Triton X-100
extraction. Accordingly, cyclin G2 and B' subunits are not just
codistributed throughout the cytoplasm like soluble cytosolic proteins
such as GFP. Rather, cyclin G2 and PP2A/B' and PP2A/B' are part
of subcellular structures resistant to Triton X-100. Our findings
indicate that cyclin G2 and PP2A/B' and - are present at the same
subcellular compartments, an important criterion for establishing their
interaction in vivo.
3
or B' were co-expressed with cyclin G2-GFP or G2-V5His for
coimmunoisolation with anti-cyclin G2 antibodies (see Fig. 3) followed
by immunoblotting with A subunit-specific antibodies. Despite the
presence of HA-tagged B'3 and B' (Fig. 5A, left
top) associated with cyclin G2 (Fig. 5A,
left bottom), we could not detect the A subunit
in these immunocomplexes (Fig. 5A, left
middle). Reciprocal immunoprecipitations with antibodies against the A subunit from corresponding cell lysates and subsequent immunoblotting with anti-cyclin G2 antibodies did not indicate any
association of cyclin G2-immunoreactive proteins with the A subunit
(Fig. 5A, right bottom). In contrast,
immunosignals for HA-tagged B' subunits present in the anti-PP2A/A
immunocomplexes were at least as strong as those for B' subunits
present in cyclin G2 precipitates (Fig. 5A, compare
right and left panels on
top), indicating that an amount of B' subunit-containing
protein complexes were immunoprecipitated that was similar if not
greater than that precipitated with anti-cyclin G2. As expected,
reprobing of the blots of anti-PP2A/A immunoprecipitates with
anti-PP2A/A antibodies yielded strong immunoreactive bands of ~62 kDa
(Fig. 5A, right middle). Although it
is impossible to rule out the presence of minor amounts of PP2A/A in
cyclin G2 complexes, these results show that a large portion of cyclin
G2-PP2A/B' complexes do not contain PP2A/A and vice versa.
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Fig. 5.
Cyclin G2 association is detectable with B'
but not A subunits. Lysates were prepared from HEK293 cells
expressing of GFP, G2-GFP, or G2-V5His and HA-tagged PP2A B' or A
subunits as indicated at the top (A) or
bottom (B) of each panel
(Transfect). A, lysates containing 500 µg of
protein were used for immunoprecipitations with anti-cyclin G2
(left panels) or rat anti-PP2A/A antibodies
(clone 6F9; right panels) and immunoblotting.
Blots were initially probed with anti-cyclin G2 (bottom
left) and/or rat anti-PP2A/A (clone 6G3; middle
right). Blots were then stripped with SDS and reprobed
in reverse order before a final stripping and reprobing with the
anti-HA antibody. HRP-coupled secondary antibodies were rabbit anti-rat
IgG and goat anti-mouse IgG for probings with anti-PP2A/A and anti-HA
antibodies, respectively. Anti-cyclin G2 antibodies were detected with
HRP-labeled protein A. B, cleared cell lysates containing 1 mg of protein were used for immunoprecipitations with anti-cyclin G2
(left panels) or rat anti-PP2A/A antibodies
(clone 6F9; right panels). Lysates containing 100 µg of protein (lanes 7-9) were also directly
loaded. Blots were probed with rat anti-PP2A/A (clone 6G3), followed by
HRP-coupled rabbit anti-rat IgG, and stripped and reprobed with
anti-cyclin G2, followed by HRP-labeled protein A.
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Fig. 6.
Cyclin G2 associates with endogenous PP2A/C
and colocalizes with PP2A/C in transfected
cells. A, cyclin G1-GST and G2-GST pulled down
endogenous PP2A/C but not PP2A/A. Lysates (500 µg of protein) were
incubated with immobilized GST, cyclin G2-GST, or G1-GST fusion
proteins for pull-down assays or were directly loaded (20 µg of
protein), as indicated. The bottom parts of each
blot were probed with mouse anti-PP2A/C followed by HRP-labeled goat
anti-mouse IgG, and top parts were probed with
rat anti-PP2A/A (clone 6G3) followed by HRP-conjugated rabbit anti-rat
IgG. B and C, cyclin G2 is associated with
endogenous PP2A/C. HEK293 cells expressing GFP, G2-GFP, and G2-V5His
with or without HA-tagged PP2A B' 3 or - as indicated were lysed.
Lysates (1 mg of protein) were used for pull-down with
microcystin-agarose (Mcys) or control resin (Sepharose
4B-CL; Seph.) (B) or for immunoprecipitations
with sheep anti-PP2A/C (C) before SDS-PAGE. Lysates (50 µg
of protein; B, lanes 1, 5,
6, 9, 10, and 13) were also
directly applied. The top half of each blot was
probed with anti-cyclin G2 followed by HRP-conjugated protein A, and
the bottom was probed with mouse anti-PP2A/C followed by HRP-labeled
goat anti-mouse IgG. The arrows indicate the position of
cyclin G2 fusion proteins and PP2A/C. D-F,
immunofluorescence confocal microscopy colocalization of PP2A/C with
cyclin G2. CHO cells were co-transfected with plasmids
encoding HA-tagged PP2A/C (D-F, left
panels; red) plus cyclin G2-V5His (D
and E, middle left
subpanels; green), G2-GFP (D and
E, middle right subpanels;
green), or GFP (F; green). Cells were
fixed either before permeabilization with Triton X-100 (D
and upper right subpanel in
F) or after pre-extraction with Triton X-100 (E
and left and lower right
subpanel of F). Cultures were stained with
anti-HA primary followed by LRSC-conjugated secondary antibodies to
visualize HA-tagged PP2A/C and TOTO-3 to label DNA (pseudocolored
blue in channel merge shown at right). Cultures
co-expressing cyclin G2-V5His were labeled with anti-cyclin G2
antibodies followed by FITC-conjugated anti-rabbit IgG.
View larger version (24K):
[in a new window]
Fig. 7.
PP2A/C directly binds to cyclin G1-GST and
cyclin G2-GST. A, HEK293 cells were transfected with
expression constructs for GFP and HA-tagged PP2A/C. Cell lysates
containing 1 mg of protein were immunoprecipitated with mouse anti-HA
antibodies. Immunocomplexes containing endogenous PP2A/A (not shown)
and HA-tagged PP2A/C were dissociated with SDS at 60 °C to obtain
immunopurified, dissociated PP2A/A and PP2A/C subunits. After
neutralization of SDS by adding an excess of Triton X-100 and removal
of the old resin, samples were used for pull-down assays with
immobilized GST, cyclin G2-GST, or G1-GST. Lysate containing 25 µg of
protein was also directly loaded for immunoblotting (lane
1). The lower part of the immunoblot
was probed with mouse anti-HA, followed by HRP-labeled goat anti-mouse
IgG. The upper part was incubated with rat
anti-PP2A/A (clone 6G3) followed by HRP-conjugated rabbit anti-rat IgG.
B-D, pull down of recombinant poly-His-tagged cyclin G2 by
immobilized recombinant PP2A/C-GST. Expression of PP2A/C-GST fusion
protein in E. coli with and without an HA tag was verified
by immunoblotting of bacterial lysates (B, lanes
1 and 2) and of glutathione-Sepharose loaded with
PP2A/C-GST, HA-tagged PP2A/C-GST, or GST alone (B,
lanes 4-6, respectively). In parallel, a lysate
of HEK293 cultures expressing HA-PP2A/C was loaded as positive control
(B, lane 3). Blots were probed with
mouse anti-PP2A/C followed by HRP-labeled goat anti-mouse IgG
(B). SDS-PAGE of 10 µl of packed beads loaded with GST,
PP2A/C-GST, and HA-PP2A/C-GST and subsequent Coomassie staining
demonstrates that comparable amounts of these proteins were present on
the beads (C). Poly-His-tagged cyclin G2 or a nonrelevant
poly-His-tagged fusion protein was expressed in E. coli, and
resulting bacterial lysates were incubated with 10 µl of each of the
three GST protein resins before immunoblotting with anti-cyclin G2
(C, lanes 5-7 and 2-4,
respectively). Bacterial lysates were also directly loaded
(C, lanes 1 and 8). Apparent molecular
masses of standards are depicted in kDa.
View larger version (37K):
[in a new window]
Fig. 8.
PP2A/C phosphatase activity is associated
with endogenous and ectopic cyclin G2 complexes. Shown is a
colorimetric assay measuring the release of phosphate following
incubation of the substrate phosphopeptide (RRA(pT)VA) with cyclin G2
or control IgG immunocomplexes isolated from CHO (A), HEK293
(B), and cerebellar (C) lysates
(graphs at left) and the corresponding
immunoblots (right panels) of indicated immunoprecipitates
isolated from the same lysates (1.5 mg (A and B,
all samples), 2 mg (C, phosphatase assay samples), and 3 mg
(C, immunoblot samples) of total protein in each lysate).
Immunoblots of respective lysates containing 30 µg (A and
B), 100 µg (C, third
lane), or 40 µg (C, sixth
lane) are shown as indicated. A, average
phosphatase activity present in cyclin G2 (n = 4) or
control IgG (n = 2) immunocomplexes isolated from
lysate of CHO cells coexpressing HA-PP2A/C with either cyclin G2V5His
or control GFP in the absence (n = 4) or presence of 20 nM okadaic acid (OA; n = 2) or 2 µM microcystin (n = 1). B,
left graph, average phosphatase activity in the presence or
absence of either 2 or 20 nM OA in the indicated
immunocomplexes isolated from lysates (in triplicate) of HEK 293 cultures transfected with either cyclin G2GFP or GFP cDNAs.
Inset graph, average phosphatase activity present in PP2A/A
(n = 3) and cyclin G2 (n = 2)
immunocomplexes isolated from 1.5 mg of lysate of the indicated cyclin
G2-expressing cultures (65% efficiency) normalized to that present in
PP2A/A immunocomplexes (100%) isolated from identical amounts of
protein lysate of cultures not expressing cyclin G2 (n = 3). C). Average phosphatase activity associated with endogenous
cyclin G2 present in anti-cyclin G2 (n = 3) or control
IgG immunocomplexes (n = 2) isolated from lysates of
rat cerebella with or without either 2 nM OA
(n = 3), 20 nM OA (n = 2),
or 2 µM microcystin (n = 1) is shown.
Shown on the right are immunoblots of lysate and anti-cyclin
G2 and anti-PP2A/C immunoprecipitates isolated from rat cerebellar
lysates, as indicated. The anti- PP2A/C immunoblot exposure times are
shorter for lane 3 relative to lanes
1 and 2 and for lanes 4 and
5 relative to lane 6 in
C.
along with either
cyclin G2GFP, G2V5, or control GFP, and the relative amount of
phosphate released in each was compared with that present in cyclin G2
immunocomplexes isolated from identical amounts of the same lysates
(Fig. 8B, inset). The phosphatase activity
associated with PP2A/A in cultures expressing cyclin G2 (65-70% of
the cells) decreased to 70-80% of that present in the PP2A/A
complexes isolated from cultures not expressing cyclin G2, whereas
cyclin G2 immunocomplexes contained 20-30% of that activity (Fig.
8B, inset). In contrast, the activity associated in PP2A/C immunocomplexes isolated from the same lysates did not vary
substantially between each condition, with similar amounts of phosphate
released whether cyclin G2 was expressed or not (data not shown). These
data suggest that although the global PP2A/C activity did not vary,
cyclin G2 at high levels competes with PP2A/A for a portion of
PP2A/C.
expression constructs indicated that the COOH terminus of cyclin G2
from amino acid 142 to 344 contains sequences that promote an
association with HA-B' (Fig. 9,
top left panels). The
NH2-terminal region from amino acids 1-140 were not
sufficient for this association. Reciprocal immunoprecipitations
followed by complementary immunoblotting showed similar results (Fig.
9, bottom left panels). These results indicate that sequences carboxyl terminal of the cyclin box are necessary and sufficient for interaction with PP2A B' subunits. Similar
coimmunoprecipitation assays examining complexes formed between PP2A/C
and GFP chimeras of truncated cyclin G2 were performed by blotting
anti-cyclin G2 immunocomplexes with antibodies to PP2A/C (Fig. 9,
right panels). Again, the results demonstrated that the carboxyl terminal region of cyclin G2 encompassing amino acids
142-344 is necessary and sufficient for interaction with PP2A but not
the NH2-terminal region encompassing the cyclin box sequences. Pull-down experiments utilizing different regions of cyclin
G2 fused to GST as immobilized targets and soluble PP2A subunits
present in mammalian cell lysates as the ligand source further verified
that the carboxyl terminus of cyclin G2 contains the sequences
necessary for interaction with PP2A (data not shown).
View larger version (67K):
[in a new window]
Fig. 9.
Determination of cyclin G2 polypeptide
regions that are necessary for both association with PP2A subunits and
inhibition of cell cycle progression. A schematic is shown of
polypeptide regions (top; the indicated numbers
represent remaining cyclin G2 residues) in NH2- or
COOH-terminal cyclin G2 deletion mutants expressed as GFP fusions. The
relative ability of the various cyclin G2 fusion proteins to interact
with PP2A/B' and C subunits in coimmunoprecipitation experiments (shown
in immunoblots below) or inhibit cell cycle progression
(quantification at bottom) is indicated by the
minus or plus signs at the
right. Left panels, anti-cyclin G2, anti-HA, or
control IgG immunocomplexes isolated from lysates of HEK293 cultures
coexpressing the indicated cyclin G2 polypeptides or control GFP with
HA-tagged PP2A/B' were probed for presence of the reciprocal protein
on indicated immunoblots (labeled at the left). Right
panels, anti-cyclin G2 or control rabbit IgG immunocomplexes
isolated from lysates of HEK293 cultures expressing HA-tagged PP2A/C
cotransfected with the indicated cyclin G2 construct or control GFP
were immunoblotted for the presence of PP2A/C (top). The
presence of comparable amounts of cyclin G2 deletion proteins
precipitated in each complex was verified by reprobing the same blots
(bottom). Bottom, tabulation of the cell cycle
distribution (see Fig. 2) of CHO cell populations expressing the
indicated GFP-linked cyclin G2 polypeptide regions or GFP-alone
controls. The shaded profiles are those shown
above to interact with PP2A subunits.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 and
B' (44) (equivalent to the rabbit B'3 and B' subunits, respectively, described by Csortos et al. (31)). Using a
variety of assays, we determined that cyclins G2 and G1 can associate with rabbit B' and with the alternative B' splice variants B'2, B'3, and B'4. An important unsolved question was if cyclin G2 or
G1 are part of the PP2A holoenzyme or act as B' subunit sinks sequestering them from the PP2A A/C core dimer and thereby perhaps altering its targeting. We show that cyclins G2 and G1 interact directly with the catalytic C subunit (Figs. 6 and 7) but not with the
scaffolding A subunit (Figs. 5 and 6). Moreover, endogenous cyclin G2
immunoisolated from rat brain cerebellum is present in a complex with
enzymatically active PP2A/C. The interactions of bacterially expressed
cyclin G2 and PP2A/C indicate that cyclin G2 can directly bind to
PP2A/C independent of both PP2A/A and B' subunits. Interaction assays
in CHO and HEK293 cells with truncated cyclin G2 mutants (Fig. 9) and
in vitro with bacterially expressed truncated cyclin G2- and
G1-GST fusion proteins4 delineated a region of ~100
residues necessary for interactions with both the catalytic C and B'
subunits. The interaction region starts at the very COOH-terminal end
of the canonical cyclin box motif. This region is not only important
for PP2A binding but also for the cyclin G2 associated cell
cycle-inhibitory effects. These results link the negative effect of
cyclin G2 expression on the cell cycle to interactions of cyclin G2
with PP2A.
or -. Additionally, due to their
somewhat similar subcellular distribution pattern when expressed alone,
changes in localization of cyclin G2 and PP2A subunits may be too
subtle to be detected in our immunocytochemical analysis of
asynchronous, unstimulated cell populations. These changes might depend
on the stage of the cell cycle, the activation of signaling pathways,
and unknown partners.
subunit to the PP2A A/C dimer that thus links certain
trimeric forms of PP2A to the cell cycle (58, 81). Nuclear localization
of PP2A/C has been described, but the exact subunit composition of the
enzyme is uncertain. Demethylated forms of PP2A/C are localized to the
nucleus and presumably deficient in binding the B subunit during the
S and G2 phases of the cell cycle (58). Many members of the
B' family carry nuclear targeting signals and are concentrated in the
nucleus (31, 42), but not all seem to require the classic bipartite
localization signal for nuclear expression (38). It is not clear if B'
subunit targeting is cell cycle position-dependent or if
demethylated forms of PP2A/C interact with B' subunits, but it has been
hypothesized that carboxylmethylation initiates a switch for A/C dimer
binding to other regulatory B subunits (81-83). Our finding that
cyclin G2 can bind to B' and C subunits and that a fraction of these
complexes are in the nucleus opens the possibility that this
interaction could directly or indirectly contribute to a change in PP2A
nuclear activity in a cell cycle-dependent manner.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: 2-530 BSB,
51 Newton Rd., Dept. of Pharmacology, University of Iowa, Iowa City, IA
52242-1109. Tel.: 319-335-8267; Fax: 319-335-8930; E-mail: mary-horne@uiowa.edu.
ABBREVIATIONS
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