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INTRODUCTION |
It is becoming increasingly clear that in addition to controlling
the cell cycle, cyclin-dependent kinases may have other functions within the cell. The CDK11 (also known as PITSLRE) protein kinases are members of the cyclin-dependent kinase
superfamily. Two distinct but closely related human CDK11 genes (Cdc2L
1 and Cdc2L 2) express several CDK11 isoforms (1, 2). CDK11 homologues exist in several species including humans, mice, chickens,
Caenorhabditis elegans, Drosophila melanogaster,
and Xenopus (3-5). The highly conserved nature of the CDK11
suggests important cellular functions.
Although CDK11 is a member of the cyclin-dependent kinase
superfamily, their function within the cell is not totally clear. However, recent studies indicate that the p110 isoform of CDK11 (CDK11p110) may be involved in some aspect of RNA
processing or transcription by virtue of the fact that CDK11
co-immunoprecipitate and/or co-purify with multiple transcriptional
elongation factors (6, 7). Furthermore, CDK11p110
associates with cyclin L (8). Cyclin L is an RS domain protein that may
function in pre-mRNA splicing (8). These observations suggest that
CDK11p110 kinases play some role in the production of
translatable RNA transcripts in proliferating cells.
The CDK11p110 isoforms contain an internal ribosome entry
site, which leads to the generation of a CDK11p58 isoform
during the G2/M phase of the cell cycle (9). Elevated expression of CDK11p58 in eukaryotic cells alters normal
cytokinesis and can delay cells in late telophase (10). In contrast,
reduced expression of CDK11p58 increases DNA replication
and enhances cell growth (11). This p58 isoform of CDK11 can interact
with cyclin D3 (12). This suggests that CDK11p58/cyclin D3
may play role in mitosis.
In regard to apoptosis, increased expression of CDK11p58
reduces cell growth due to apoptosis (1). In addition, our group and
others have shown that the CDK11p110 isoform and the
CDK11p58 isoform are cleaved by caspases to generate a
smaller 46-50-kDa protein that contains the catalytic portion of the
protein (2, 13, 14). This smaller CDK11p46 isoform can be
triggered by Fas, tumor necrosis factor A, or staurosporine and
phosphorylate histone H1. Caspase inhibitors can modulate the kinase
activity of the caspase-processed isoform of CDK11 (2). Collectively,
these observations suggest that CDK11 may play a role in apoptotic
signaling. However, substrates potentially regulated by CDK11 during
apoptosis have not been identified. The present study was performed to
address this issue. We identify the p47 subunit of eukaryotic
initiation factor 3 (eIF31
p47) as a protein associated with the caspase-processed isoform of
CDK11 (CDK11p46) using a yeast two-hybrid screen. The
interaction between CDK11p46 and eIF3 p47 occurs in
vitro and in vivo. In addition, CDK11 protein kinase
isolated from cells undergoing apoptosis can phosphorylate eIF3 p47
in vitro, and serine phosphorylation of eIF3 p47 occurs in
cells during apoptosis. Taken together, these results strongly support
our hypothesis that CDK11 may be involved in apoptotic signaling by
interacting with eIF3 p47.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Treatments--
A375 human melanoma cell line
was obtained from American Type Culture Collection (Manassas, VA). The
cells were cultured at 37 °C with 5% CO2 in RPMI 1640 medium (Mediatech, Inc., Herndon, VA), supplemented with 5% fetal
bovine serum (Omega Scientific, Inc., Tarzana, CA), 1%
L-glutamine, and 1% penicillin/streptomycin (Invitrogen).
For induction of apoptosis, cells were treated with either 0.5 µg/ml
anti-Fas antibody (Upstate Biotechnology, Inc., Lake Placid, NY) or 10 ng/ml staurosporine (Sigma) from the second day of seeding for the
indicated time. HEK293 (human embryonic kidney 293) cell line was
cultured in Dulbecco's modified Eagle's medium (Mediatech)
supplemented with 10% fetal bovine serum, 1% L-glutamine,
and 1% penicillin/streptomycin at 37 °C with 5% CO2. All transfections were carried out using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions.
Antibodies--
GN1 is an affinity-purified rabbit polyclonal
antibody raised by injection of rabbit with purified recombinant
glutathione S-transferase (GST) fused with the sequence
coding for amino acids 341-413 of CDK11p110 (Rockland,
Gilbertsville, PA). CDK11 monoclonal antibody P1C recognizes the last
75 amino acids of CDK11p110 and was a gift from Drs.
Vincent Kidd and Jill Lahti (St. Jude Children's Research Hospital,
Memphis, TN) (7). EIF3 p47 antibody is a polyclonal antibody raised by
injection of goat with purified recombinant GST-fused full-length eIF3
p47 (Rockland). eIF3 antibody was raised in a goat as described (15).
Rabbit polyclonal HA antibody, monoclonal c-Myc antibody and monoclonal
anti-phosphoserine antibody were purchased from Sigma.
Yeast Two-hybrid Assay--
Matchmaker GAL4 two-hybrid system 2 (Clontech, Palo Alto, CA) was used to perform yeast
two-hybrid screening according to the manufacturer's instructions. The
wild type and K451M substituted CDK11 C-terminal portion
(CDK11p46) were used as baits. In the K451M altered
CDK11p46, one lysine residue that is associated with
phosphate transfer was substituted with a methionine. This substitution
was performed to stabilize the interaction between the kinase and its
substrate (16). A total of 5 × 106 transformants from
a human fetal brain Matchmaker cDNA library (Clontech) were screened in the yeast strain AH109
(Clontech), and 28 colonies were identified as
His+Ade+, out of which 13 were positive for
-galactosidase. Among the 13 positive clones, 12 were isolated when
the K451M mutant CDK11p46 was used as bait, whereas one
clone was isolated using wild type CDK11p46 as bait. One of
these clones (clone 4) was revealed to encode the full-length cDNA
to the p47 subunit of eukaryotic translation initiation factor 3 gene
by sequence analysis. To confirm the CDK11p46/eIF3 p47
interaction in yeast, 0.1 µg of clone 4, and wild type bait plasmid
were co-transfected into yeast strain AH109 using the lithium acetate
transformation method. Growth selection was performed according to the
manufacturer's protocol (Clontech).
Construction of CDK11p46
Vectors--
pCMV-Myc-CDK11p46 containing the
CDK11p46 coding sequence (GenBankTM accession
number U04824; nucleotides 1282-2465) with a c-Myc tag at the N
terminus was constructed. CDK11p46 was generated by PCR
using p110KS+ (a gift from Dr. Vincent Kidd) as template.
pGEX-4T-2-CDK11p46 that produces a GST fusion
CDK11p46 in bacteria was constructed by inserting the same
PCR product of CDK11p46 in frame into the EcoRI
site of pGEX-4T-2 vector (Amersham Biosciences). pcDNA3-CDK11p46 was constructed by cloning the PCR
amplified CDK11p46 with an in frame start codon ATG
(nucleotides 1121-2465) into the EcoRI site of a eukaryotic
expression vector pcDNA3 (Invitrogen) for stable transfection purposes.
Construction of eIF3 p47 Vectors--
pCMV-HA-eIF3 p47,
containing the full-length coding sequence to the eIF3 p47 with a HA
tag at the N terminus, was constructed. Full-length eIF3 p47 was
isolated from the original fetal brain cDNA clone we obtained by
yeast two-hybrid assay using EcoRI/XhoI restriction enzymes. The full-length eIF3 p47 was cloned into pGEX-4T-2
to generate GST-eIF3 p47 fusion protein expressed in E. coli. Four DNA fragments encoding truncated eIF3 p47, amino acids
106-360, 170-360, 248-360, and 1-113 were obtained by PCR amplification and cloned into pGEX-4T-2 vector to generate deletion GST-p47 fusion proteins.
Construction of CDK11p46 Mutants--
The K451R
mutation was generated by polymerase chain reaction using a
QuikChangeTM site-directed mutagenesis kit (Stratagene, La
Jolla, CA) with CDK11p46 wild type construct as template.
Sequencing analysis was used to confirm the mutation. This mutation has
been shown to cause CDK11 to lose its kinase activity (1). Therefore,
we created this mutation in the ATP binding domain to inactivate the
kinase. The mutagenesis primers were
5'-GAAATTGTGGCTCTAAGGCGGCTGAAGATGG-3' and
5'-CCATCTTCAGCCGCCTTAGAGCCACAATTTC-3'.
Immunoprecipitation and Western Blotting--
Cells were
harvested, washed twice with cold PBS, and lysed in lysis buffer (10 mM Hepes, pH 7.2, 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2% Nonidet
P-40) containing 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1% protease
inhibitor mixture (Sigma) for 30 min on ice. Following lysis, cells
were centrifuged at 13,000 × g for 10 min at 4 °C,
and the protein content was determined using the bicinchoninic acid
assay (Pierce). Total cell lysate (500 µg) was precleared with
protein A- or G-agarose beads (Oncogene, La Jolla, CA) and goat IgG
(Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 1 h.
Myc-CDK11p46 fusion protein or eIF3 was then
immunoprecipitated using c-Myc monoclonal antibody (Sigma) or goat eIF3
polyclonal antibody (15) and protein A- or G-agarose overnight at
4 °C. The immune complex was then washed three times with lysis
buffer and subjected to SDS-PAGE. The proteins were transferred to a
polyvinylidene difluoride membrane (Bio-Rad), and the blots were probed
with different antibodies. A secondary probe with horseradish
peroxidase-labeled antibodies (Sigma) was detected by enhanced
chemiluminescence (ECL) (Amersham Biosciences).
Immunofluorescence Confocal Microscopy--
pCMV-HA-eIF3 p47
transiently transfected A375 cells were grown on coverslips, washed
twice with cold PBS, and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were rinsed three times with PBS and
permeabilized with 100% methanol at
20 °C for 6 min. Cells on
coverslips were washed with PBS again and incubated with 5% bovine
serum albumin in PBS for 10 min at room temperature, and then the
bovine serum albumin was removed. The coverslips were then incubated
with CDK11p110-specific antibody P1C (1:500 dilution) and
rabbit anti-HA antibody (1:50 dilution) for 1 h at room
temperature. Coverslips were washed three times with PBS for 5 min each
and then incubated with fluorescence-labeled secondary antibodies
fluorescein isothiocyanate-conjugated anti-mouse IgG + IgM (1:100
dilution) and Cy5-conjugated anti-rabbit IgG (1:100 dilution) in
darkness for 1 h at room temperature. Following incubation,
coverslips were washed three times with PBS, mounted with DAKO mounting
medium, and stored at 4 °C overnight for immunofluorescence confocal microscopy analysis.
Cell Fractionation--
A375 cells were washed with PBS and
resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) containing 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1%
protease inhibitor mixture (Sigma) and incubated on ice for 15 min.
Cells were then homogenized using the Dounce tissue grinder (Wheaton,
Millville, NJ) with 25-30 strokes, and the percentage of nuclei was
checked by trypan blue staining. Nuclei were collected at 2000 rpm for
10 min. The supernatant contained the cytoplasm.
Purification of Recombinant Protein from E. coli--
GST,
GST-CDK11p46, GST- CDK11p46M, and GST-p47 were
induced by 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside and expressed in
BL21 bacteria for 4 h at 30 °C. The recombinant proteins were
purified using Bulk GST Purification Module according to the
manufacturer's instruction (Amersham Biosciences). Purified proteins
were concentrated using Centricon 30 (Amicon Inc.) to an appropriate
concentration and stored as aliquots at
70 °C.
GST Pull-down Assay--
The assay was performed as described
previously (12) with a few modifications. Briefly, GST or GST fusion
proteins were expressed in BL21 cells, and equal amounts of bacterial
lysates were incubated with 25 µl of glutathione-Sepharose beads for
30 min. The beads were then washed three times with PBS and incubated with 5 µl of in vitro transcribed and translated
[35S]methionine-labeled CDK11p46 overnight at
4 °C. [35S]Methionine-labeled CDK11p46 was
produced using a TNT-coupled reticulocyte lysate system (Promega). The
beads were then washed five times with binding buffer and boiled in SDS
sample buffer. The bound CDK11p46 protein was analyzed by
autoradiography after resolved by SDS-PAGE.
Kinase Assay--
A375 cells were treated with anti-Fas (Upstate
Biotechnology) or with both anti-Fas and caspase-3 inhibitor DEVD-FMK
(50 µM) for 0, 24, 36, and 48 h and harvested, and
kinase assays were carried out as described (2). Lysates of A375 cells
stably transfected with kinase mutant CDK11p46M were also
used in the kinase assays. 5 µg of either GST or GST-eIF3 p47 were
used as substrates for kinase assays. Phosphorylation of the substrates
was analyzed by 12% SDS-polyacrylamide gel electrophoresis and
autoradiography. The gel was exposed overnight at room temperature. Amersham Biosciences ImageQuant software was used to quantitate the relative phosphorylation level of the substrates.
Identification of eIF3 p47 Phosphorylation
Site--
Caspase-processed CDK11p110 was
immunoprecipitated from anti-Fas-treated A375 cells. Recombinant eIF3
p47 was then phosphorylated by caspase-processed CDK11p110
as previously described (2) and was separated by SDS-PAGE. The
recombinant eIF3 p47 was excised from the gel, digested with pepsin,
and analyzed for phosphoamino acids by mass spectrometry as previously
described (17, 18) by the Arizona Cancer Center Proteomic Core Facility.
In Vitro Translation Inhibition Assay--
First, a time course
study and a dose response study were performed to determine the linear
range of luciferase production using the rabbit reticulocyte lysate
system. 50 ng of luciferase mRNA were added into the system and
incubated at 30 °C for 0-120 min followed by measurement of
luciferase activity every 15 min to determine the luciferase production
rate over the time course. The production of luciferase is in a linear
range up to 75 min of incubation. 20-50 ng of luciferase mRNA were
incubated with the system at 30 °C for 60 min followed by
measurement of luciferase activity. The production of luciferase is in
a linear range between 20 and 50 ng of luciferase mRNA. Therefore,
40 ng of firefly luciferase mRNA was added to 50 µl of a rabbit
reticulocyte lysate in vitro translation reaction (Promega,
Madison, WI) in the presence of recombinant purified 18-70
nM of CDK11p46, CDK11p46M, 70 nM GST, or elution buffer. The reaction mixtures were
incubated at 30 °C for 1 h. Luciferase activity was measured
with a luminometer. In addition, newly synthesized
35S-labeled luciferase proteins were analyzed by subjecting
equal amounts of reaction mixtures to 10% SDS-PAGE and autoradiography.
In Vivo Reporter Synthesis Assay--
25 ng of pGL3
control vector (Promega) containing SV40 promoter-driven luciferase
gene was transfected into A375 cells that were stably transfected with
either pcDNA3 (A375-pcDNA3), pcDNA3-CDK11p46M
(A375-CDK11p46M), or pcDNA3-CDK11p46
(A375-CDK11p46) in 24-well plates in triplicate. After
24 h, cells were lysed in passive lysis buffer (Promega), and
luciferase activity was measured using the luciferase reporter assay
system (Promega).
RT-PCR and Quantitative Real Time One-step RT-PCR--
Total
RNAs were extracted from A375-pcDNA3 and A375-CDK11p46
cells transfected with pGL3 control vector and A375 cells untransfected with pGL3 in triplicate using RNeasy Mini Kit (Qiagen, Valencia, CA).
Reverse transcription was performed with 2 µg of total RNA using the
Omniscript RT kit (Qiagen). 2 µl of the cDNA mix was added to 20 µl of the PCR containing 2.5 units of Taq polymerase (TaKaRa, Otsu, Shiga, Japan), 1× buffer, and 1 pmol of primers. For
histone, PCR was carried out at 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C
for 45 s with a final extension at 72 °C for 5 min using the
primers Histone-F (5'-CCACTGAACTTCTGATTCGC-3') and Histone-R
(5'-GCGTGCTAGCTGGATGTCTT-3'). For luciferase, PCR was carried out at
95 °C for 5 min followed by 30 cycles at 95 °C for 1 min,
65 °C for 1 min, and 72 °C for 1 min with a final extension at
72 °C for 10 min using the primers LucF (5'-AATTGCTTCTGGTGGCGCTC-3')
and LucR (5'-GGGGTGTTGGAGCAAGATGG-3'). The amplified fragments were
analyzed by 1% agarose gel electrophoresis and stained with ethidium
bromide. Real time RT-PCR was performed using SYBR® Green PCR Core
Reagents (PerkinElmer Life Sciences) and Omniscript RT kit (Qiagen) and
amplified in a PerkinElmer Life Sciences prism 5700 sequence detection
system according to the manufacturer's instructions. Dissociation
curves were performed to determine the specificity of the amplicon.
Each sample was completed in triplicate, and no template control was
included. Threshold cycle (Ct) during the exponential phase of
amplification was determined by real time monitoring of fluorescent
emission. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
a control gene. Primers for GAPDH were purchased from PerkinElmer Life
Sciences. Primers for luciferase are
5'-CGTCTTAATGTATAGATTTGAAGAAG-3' and
5'-TGGCGAAGAAGGAGAATAG-3'. Luciferase mRNA levels were
demonstrated as the absolute number of copies normalized against GAPDH
mRNA. Difference in amplification was indicated as 1/(Ct
luciferase
Ct GAPDH) (19, 20).
Overall Cellular Protein Synthesis Inhibition Assay--
A375 or
A375-CDK11p46 cells were plated at 2 × 104 cells/well in 96-well plates in RPMI 1640 medium
without leucine (U.S. Biological, Swampscott, MA) in triplicate and
incubated at 37 °C for the indicated time. Cells were either treated
with ethanol or treated with 10 ng/ml of staurosporine for the
indicated time. 6 h before the end of the incubation, 1 µCi of
L-[3,4,5-3H]leucine (PerkinElmer Life
Sciences) per well was added. The cells were collected onto UniFilter
by cell harvester, and the incorporated [3H]leucine was
measured by a microplate scintillation counter (Packard, Meriden, CT).
Apoptosis and Cell Survival Assays--
Cells were stained with
7-aminoactinomycin D (7-AAD) and analyzed for apoptosis using a FACStar
flow cytometer (Becton Dickinson, San Jose, CA) as described (2). Cell
morphology analysis was performed by cytospin preparation of cells
followed by methanol fixation for 6 min. Slides were air dried and
stained for 15 min in Giemsa stain. Slides were then rinsed in
deionized water and air-dried followed by light microscopy analysis at
×15 magnification. Cell viability was measured with a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay as described (2).
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RESULTS |
The Kinase Domain of the CDK11 Protein Kinase
(CDK11p46) Interacts with the Mov34/JAB_MPN
Domain of eIF3 p47--
The yeast two-hybrid method was used to screen
a human fetal brain cDNA library using the caspase processed
C-terminal portion containing the kinase domain of
CDK11p110 (CDK11p46) as bait, and 13 CDK11p46-interacting cDNA clones were isolated. DNA
sequencing and data base searching revealed that the nucleotide
sequence of clone 4 encoded the full-length human p47 subunit of
eukaryotic initiation factor 3 (eIF3 p47). The specificity of the
interaction was confirmed by co-transformation of the bait and clone 4 again into yeast AH109, and various positive controls and negative
controls were included in the nutritional deficiency selection.
Characterization of the other 12 clones is in progress in our laboratory.
The interaction of CDK11p46 with eIF3 p47 was further
tested in vitro using a GST pull-down assay. The in
vitro translated 35S-labeled CDK11p46
could directly and specifically bind to GST-p47 eIF3 fusion protein (Fig. 1B). EIF3 p47 is a Mov34
family member. The function of the Mov34/JAB_MPN domain in eIF3 p47 has
not been established. We speculated that Mov34/JAB_MPN related domain
in eIF3 p47 might be involved in protein-protein interactions. To
determine the region in eIF3 p47 interacting with CDK11p46,
we deleted specific sequences within eIF3, made fusion proteins, and
performed GST pull-down assays (Fig. 1A). In the GST
pull-down assay, in vitro translated 35S-labeled
CDK11p46 protein could be pulled down by GST-p47 amino
acids 106-360 and GST-p47 amino acids 170-360 but not by GST-p47
amino acids 248-360 or GST-p47 amino acids 1-113 (Fig. 1,
A and B). This suggests that the region within
eIF3 p47 that directly interacts with CDK11p46 is between
amino acids 113 and 248, which contains the Mov34/JAB_MPN domain.
Therefore, the interaction of the caspase-processed isoform of CDK11
(CDK11p46) with eIF3 p47 may be mediated through the
Mov34/JAB_MPN domain in vitro, and the major region of
interaction appears to be the C-proximal half of the Mov34 domain.

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Fig. 1.
The kinase domain of CDK11p110
(CDK11p46) interacts with the Mov34/JAB_MPN domain of eIF3
p47. A, the top diagram
illustrates full-length eIF3 p47 and the location of the Mov34/JAB_MPN
domain. Four GST-p47 truncation mutants were designed to localize the
binding site of p47 with the kinase domain of CDK11p110.
One-letter amino acid codes are shown. B, the
35S-labeled in vitro transcribed and translated
kinase domain of CDK11p110 (CDK11p46) was
incubated with GST, full-length GST-p47, and the deletion mutants of
p47 in a GST pull-down assay. The bound CDK11p46 was
separated by SDS-PAGE and visualized by autoradiography.
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To further demonstrate the interaction in human cells, an expression
vector with an HA epitope-tagged full-length eIF3 p47 was constructed
and transfected into A375 cells. Immunofluorescence confocal microscopy
using an anti-HA antibody revealed that p47 localized both in
nucleoplasm and cytoplasm, especially the region around
nucleus (Fig. 2A).
CDK11p110 localizes predominantly to the nucleoplasm as
revealed by P1C antibody. No fluorescence was seen with the HA antibody
in untransfected cells (data not shown). Merging of the two images
showed a yellow color in the nucleoplasm (Fig. 2A),
indicating that a portion of eIF3 p47 co-localizes in the nucleus with
CDK11. To confirm this observation, we performed cell fractionation and
Western blot analysis. Again, we observed both a cytoplasmic and
nuclear pool of eIF3 p47, whereas CDK11 was seen in the nucleus (Fig. 2B). Thus, CDK11 and a portion of eIF3 p47 can both be found
in the nucleus. In addition, reciprocal immunoprecipitation and Western analysis with Myc-tagged CDK11p46 and HA-tagged eIF3 p47
from transfected HEK293 cells showed that CDK11p46 and eIF3
p47 could associate in cells (Fig. 2C). These results together with the following endogenous interaction data suggest that
CDK11p46 interacts with eIF3 p47 in human cells.

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Fig. 2.
The co-localization of CDK11 and eIF3 p47 in
human cells. A, co-localization of
CDK11p110 and eIF3 p47 in the nucleus. A375 cells were
transfected with pCMV-HA-p47, and 24 h post-transfection, a
confocal immunofluorescence microscopy assay was performed using P1C
antibody and anti-HA antibody. The subcellular location of
CDK11p110 (green) and eIF3 p47
(red) and their co-localization (yellow)
are shown (magnification, ×100). B, localization of
CDK11p110 and eIF3-p47 by cell fractionation and Western
blot. 20 µg of fractionated A375 cell lysates were used for each lane
and CDK11p110-specific antibody GN1 or anti-eIF3 antibody
was used for immunoblotting. C, the interaction between
transfected CDK11p46 and eIF3 p47 in human cells. HEK293
cells were cotransfected with pCMV-Myc-CDK11p46 and
pCMV-HA-p47. 48 h post-transfection, cells were harvested, and
cell extracts were made. Immunoprecipitation was performed with either
mouse IgG or anti-Myc antibody followed by Western blotting with
anti-Myc or anti-HA antibody.
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Endogenous eIF3 p47 Interacts with CDK11 during
Apoptosis--
Next we tested whether p47 is a potential
substrate of CDK11 during apoptosis. During apoptosis, the p110 isoform
of CDK11 is cleaved by caspase-3 into a p46-50 C-terminal fragment
containing the kinase domain and a p60 N-terminal fragment (2). We saw a weak interaction between the endogenous CDK11p110 isoform
and eIF3 in untreated A375 cells (Fig. 3,
lane 6). Interestingly, when apoptosis is induced
by anti-Fas, the caspase-processed C-terminal fragment of
CDK11p110 interacted strongly with eIF3. The interaction
was strongest at 36 h after stimulation of apoptosis (Fig. 3,
lanes 7-9). The specificity of the interaction
was confirmed by including nonspecific goat IgG as negative controls
(Fig. 3, lanes 1-4), and the correct sizes of
the expected protein bands are shown in lanes 5,
10, and 11 (Fig. 3). In CDK11p46
stable transfected cells, the interaction between eIF3 and
CDK11p46 (p46-p50) was strong (Fig. 3, lane
5), which means that the overexpressed CDK11p46
associates with eIF3, presumably through its p47 subunit. In order to
prove that the cells did undergo apoptosis after anti-Fas or
staurosporine treatment during the time course, the percentage of
apoptotic cells was measured by flow cytometry (Fig.
4A), and the cell viability
was analyzed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay (Fig. 4B). In addition, apoptosis was confirmed by
light microscopy analysis (data not shown). The results indicated that
the cells underwent apoptosis gradually after stimulation and the
percentage of apoptotic cells approached 100% by 72 h after
treatment (Fig. 4A). These observations are consistent with the previous findings by our group (2).

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Fig. 3.
The interaction between endogenous
CDK11p46 and eIF3 p47 during apoptosis. A375 cells
were treated with 0.5 µg/ml anti-Fas antibody to stimulate apoptosis
for 0, 24, 36, and 48 h, and then the cells were lysed and used
for immunoprecipitation. CDK11p46 overexpressed A375 cells
were used in lanes 5 and 11. In
lanes 1-4, immunoprecipitation was performed
with goat anti-GST IgG, whereas in lanes 5-9,
immunoprecipitation was performed with goat anti-eIF3 antibody followed
by immunoblotting with CDK11p110-specific antibody, GN1. 20 µg of cell lysates were used in lanes 10 and
11 for Western blot.
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Fig. 4.
Time course study of apoptosis induced by
anti-Fas antibody or staurosporine. A, A375 cells were
treated with 0.5 µg/ml of anti-Fas antibody for 0, 24, 36, 48, and
72 h and analyzed for apoptosis by 7-AAD staining followed by flow
cytometry. The y axis indicates the percentage of total
apoptotic cells. B, A375 cells were treated with 10 ng/ml
staurosporine for 0, 24, 48, and 72 h, and cell viability was
measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay. *, p < 0.05.
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CDK11p46 Phosphorylates eIF3 p47 during Apoptosis in
Vitro--
Since CDK11p46 contains the kinase domain of
CDK11 and it has been shown by our group and others that the kinase
activity of CDK11 is stimulated during apoptosis (1, 2), we
hypothesized that CDK11p46 may phosphorylate eIF3 p47
during apoptosis. CDK11 was immunoprecipitated from anti-Fas-treated
A375 cell lysates, and GST control or GST recombinant p47 protein were
used as substrates in kinase assays. Immunoprecipitated CDK11 from A375
cells undergoing apoptosis was capable of phosphorylating recombinant
eIF3 p47 protein (Fig. 5A). In
contrast, CDK11p46 did not phosphorylate GST alone.
Furthermore, the addition of a caspase-3 inhibitor (DEVD-FMK)
diminished the degree of eIF3 p47 phosphorylation (Fig. 5B,
lane 3), and A375 cells stably transfected with
kinase mutant CDK11p46M displayed suppressed
phosphorylation of eIF3 p47 (Fig. 5B, lane 4). These results indicate that the caspase-processed
isoform of CDK11p110 isolated from cells undergoing
apoptosis phosphorylates eIF3 p47 in vitro. The results with
the specific caspase-3 inhibitor and the mutant form of
CDK11p46 kinase indicate that the phosphorylation of eIF3
p47 is due to the CDK11p46, not to some other kinase that
copurifies in the immunoprecipitate.

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Fig. 5.
EIF3 p47 is phosphorylated by the
caspase-processed CDK11p110 isoform during apoptosis.
A, A375 cells were treated with 0.5 µg/ml anti-Fas
antibody or 10 ng/ml staurosporine to stimulate apoptosis for 0, 24, 36, 48 h, and then the cells were harvested and lysed. 300 µg of
each cell lysate was used for immunoprecipitation using
CDK11p110-specific antibody GN1 and protein A-agarose. The
precipitate was then incubated with 5 µg of the substrate GST or
GST-fused eIF3 p47 (GST-p47) in the presence of
[ -32P]ATP in kinase buffer followed by SDS-PAGE and
autoradiography or phosphorimaging to quantitate the incorporated
32P. The figure shows a representative
result from at least three independent experiments. B, A375
cells were treated with 0.5 µg/ml anti-Fas antibody and/or 50 µM caspase-3 inhibitor (DEVD-FMK) for 36 h. In
lane 4, A375 cells stably transfected with kinase
mutant CDK11p46M were used. Cells were then lysed for the
kinase assay as described for A. 5 µg of GST-p47 were used
as substrate.
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Identification of the Phosphorylation Site in eIF3 p47 and the in
Vivo Phosphorylation Status of p47 during Apoptosis--
To determine
the phosphorylation site in the eIF3 p47 protein phosphorylated by
CDK11, recombinant eIF3 p47 was incubated with caspase-processed CDK11
that immunoprecipitated from anti-Fas-treated cell lysate in kinase
buffer. After the kinase reaction, recombinant eIF3 p47 protein was
separated by SDS-PAGE. EIF3 p47 was digested with pepsin in gel and
subjected to liquid chromatography/MS/MS analysis. The spectrum for the
p47 phosphopeptide (residues Ala-41 to Pro-49),
[M-H3PO4] = 784 m/z is
shown in Fig. 6A.
Identification of a phosphorylated Ser46 is seen by
analysis of b ions and y ions. A gain of
phosphate at the y4 ion, 485 m/z,
derived from fragmentation between Ser-45 and Ser-46 was observed. The
reason that y ions are not completely shown is that multiple
internal cleavages instead of sequential cleavages from the carboxyl to
amino terminus occurred. Once we found that Ser-46 within eIF3 p47 was
phosphorylated by CDK11 in vitro, we investigated the serine
phosphorylation status of eIF3 p47 in cells undergoing apoptosis
in vivo. A375 cells were treated with staurosporine to
trigger apoptosis, and cell lysates were immunoprecipitated with
eIF3-specific antibody followed by Western analysis with
phosphoserine-specific antibody or eIF3 p47-specific antibody. In A375
cells stimulated to undergo apoptosis, serine phosphorylation of
eIF3 p47 was seen, and the maximum phosphorylation level was seen at
36 h after treatment (Fig. 6B). These results are
consistent with our in vitro kinase assay and show that
serine phosphorylation of eIF3 p47 occurs during apoptosis in
vivo.

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Fig. 6.
Determination of the phosphorylation site in
eIF3 p47 by the caspase-processed CDK11p110 isoform and
characterization of serine phosphorylation during apoptosis in
vivo. A, MS identification of the eIF3-p47
in vitro phosphorylation site. CDK11p110 was
immunoprecipitated from anti-Fas-treated A375 cell lysate using
CDK11p110-specific antibody GN1 and incubated with
recombinant GST-p47. Then an in-gel pepsin digest was carried out. MS
spectra of the identified serinic-phosphopeptide showed that Ser-46 is
the site of phosphorylation. Annotated ions are shown as nominal
m/z values and resulted from fragmentation of an
amide bond with charge retention on the carboxyl (y ions) or
amino terminus (b ions). B, serine
phosphorylation of eIF3-p47 during apoptosis in vivo. A375
cells were treated with staurosporine for 0, 24, 36, 48, and 72 h.
Cell lysates were immunoprecipitated with eIF3 antibody and then
immunobloted with phosphoserine antibody or eIF3 p47-specific
antibody.
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CDK11p46, but not the Kinase Mutant
CDK11p46M, Inhibits in Vitro Translation--
We further
investigated the functional consequences of the association of
caspase-processed CDK11p110 with eIF3 p47. We
determined the effect of CDK11p46 on protein
synthesis in vitro. A rabbit reticulocyte in
vitro translation system was used for this purpose. In this
system, a given mRNA is translated into protein. For our studies,
we choose to use the luciferase gene product for two reasons. First, we could monitor the luciferase activity as a measurement of protein production, and second, incorporation of radioactive
[35S]methionine into the synthesized protein can act as a
different measurement of protein production. We established that our
assay conditions were within the linear range. This was achieved by measuring luciferase activity every 15 min throughout the incubation and adding a defined amount of luciferase mRNA (40 ng) into the system. We found that the rate of luciferase production is within the
linear range between 0 and 75 min of incubation time (Fig. 7A) (R2 = 0.9906, p < 0.01). Next, we investigated whether the
dose response of luciferase mRNA to translation in vitro
is linear. We found that adding luciferase mRNA between 20 and 50 ng is within the linear range (Fig. 7A)
(R2 = 0.9791, p < 0.01).
Therefore, we chose 40 ng of luciferase mRNA and incubated for 60 min in subsequent experiments. Luciferase mRNA was translated in
the presence or absence of added recombinant CDK11p46 or
CDK11p46M protein. Both luciferase activity and
incorporated [35S]methionine were measured.
CDK11p46 significantly inhibited the synthesis of
luciferase (Fig. 7B). The specificity of this observation
was verified by adding GST, another purified Escherichia
coli expressed protein, to the reaction, and it had no effect on
translation compared to the buffer (Fig. 7B, top
panel). This observation was further confirmed by performing a dose-response assay with recombinant CDK11p46 protein. A
concentration-dependent inhibitory effect of
CDK11p46 protein on translation was observed (Fig.
7B). Moreover, the phosphorylation of eIF3 p47 by
CDK11p46 appears to be required for the inhibition of
translation, since the kinase-defective mutant CDK11p46M
did not inhibit the synthesis of luciferase (Fig. 7B).

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Fig. 7.
Inhibition of translation by
CDK11p46 but not kinase mutant CDK11p46M
in vitro. A, the rate of
luciferase production and the dose response to luciferase mRNA were
determined. 50 ng of luciferase mRNA were translated in
vitro for 15-120 min, and luciferase activity was then measured
with a luminometer. Twenty to fifty nanograms of luciferase mRNA
were translated in vitro, and luciferase activity was
measured. The incubation time or ng of luciferase mRNA was plotted
against luciferase activity. B, CDK11p46, but
not kinase mutant CDK11p46M, inhibits translation in a
dose-dependent manner in vitro. Luciferase
mRNA (40 ng) was translated in an in vitro translation
system in the presence of 18, 35, or 70 nM recombinant
purified CDK11p46 or CDK11p46M protein or 70 nM purified recombinant GST. Luciferase protein synthesis
was analyzed by gel electrophoresis followed by autoradiography.
Luciferase activity was measured with a luminometer. The averages of
results from three experiments are shown. *, p < 0.05.
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CDK11p46, but Not Kinase Mutant CDK11p46M,
Inhibits Translation in Vivo--
To examine the ability of
CDK11p46 to inhibit translation in human cells, a
luciferase reporter plasmid was transiently transfected into A375 cells
stably transfected with either pcDNA3, CDK11p46, or
kinase-defective mutant CDK11p46M. Luciferase activity was
then measured 24 h after transfection. Luciferase synthesis was
significantly inhibited in A375 cells that overexpress
CDK11p46 compared with the vector-transfected cells (Fig.
8A, left
panel). In contrast, luciferase activity was slightly
elevated in the CDK11 kinase-defective mutant
CDK11p46M-transfected cells relative to vector-transfected
cells (Fig. 8A, right panel). To rule
out the effect of CDK11p46 on transcription of the
luciferase gene, RT-PCR was performed on three different transfected
clones from either vector alone- or luciferase reporter
gene-transfected cells. The histone gene served as a control (246 bp).
The luciferase mRNA of vector- or CDK11p46-transfected
cells was the same (Fig. 8B, 454 bp), indicating that
CDK11p46 inhibited the translation of the luciferase
reporter gene and not transcription. To further quantify the luciferase
mRNA, real time PCR was performed from pcDNA3 vector- or
CDK11p46-transfected cells. The Ct of luciferase was
normalized against GAPDH. No difference was observed between vector-
and CDK11p46-transfected A375 cells (Fig. 8C).
Untransfected A375 cells and no template control served as negative
controls (Fig. 8C). These results suggest that the
caspase-processed CDK11p110 isoform inhibits protein
synthesis in vivo at the translational level.

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Fig. 8.
Inhibition of the synthesis of
luciferase reporter gene by CDK11p46 but not kinase mutant
CDK11p46M in vivo.
A, CDK11p46, not CDK11p46M, inhibits
translation of the luciferase reporter gene in vivo.
Triplicate pcDNA3-, pcDNA3- CDK11p46-, or
pcDNA3-CDK11p46M-transfected cells were
transfected with pGL3-SV40, which contains a luciferase reporter gene.
24 h post-transfection, cells were lysed, and luciferase activity
was measured by a luminometer. *, p < 0.05;
****, p < 0.0001. B, CDK11p46
did not inhibit transcription of the reporter gene. pcDNA3- or
pcDNA3-CDK11p46-transfected cells were transfected with
pGL3-SV40, which contains a luciferase reporter gene. After 24 h,
cells were harvested, and total RNA was isolated for RT-PCR analysis.
The expected PCR products for histone and luciferase are 246 and 454 bp, respectively. Three different clones of each transfected cells were
tested. C, quantitative real time PCR also indicates that
there is no difference in luciferase mRNA level between vector- and
CDK11p46-transfected cells. The same RNA samples were
analyzed for luciferase transcripts by one-step real time RT-PCR.
Luciferase mRNA levels were normalized against GAPDH and
illustrated as mean ± S.D. NTC, no template control;
Luc /Luc+, luciferase reporter gene
untransfected/transfected cell lines.
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CDK11p46 Suppresses Overall Protein Synthesis--
To
further investigate the impact of CDK11p46 on overall
cellular protein synthesis, we established two stably transfected cell lines. One was stably transfected with pcDNA3 vector, and the other
was stably transfected with CDK11p46. Overall protein
synthesis, as measured by pulse labeling with [3H]leucine
in vivo, was significantly lower in
CDK11p46-overexpressing cells compared with
vector-transfected cells (Fig. 9A). Therefore,
CDK11p46 can also inhibit the overall rate of cellular
protein synthesis. Since it was reported that the rate of protein
synthesis is rapidly down-regulated in mammalian cells following the
induction of apoptosis (21-24), we also measured protein synthesis in
A375 cells after staurosporine treatment. Consistent with these
findings, the overall cellular protein synthesis decreased during
apoptosis (Fig. 9B).

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Fig. 9.
Inhibition of overall protein synthesis by
CDK11p46 in vivo. A,
CDK11p46 inhibits overall cellular protein synthesis
in vivo. Cells stably expressing CDK11p46
or neomycin were plated in 96-well plates and incubated in medium
without L-leucine for 24 h. 6 h before
harvesting, L-[3H]Leucine was added to the
medium, and the incorporated L-[3H]leucine
was measured with a scintillation counter. ***, p < 0.001. B, the overall protein synthesis decreases during
apoptosis. Cells were incubated in medium without L-leucine
for 24 h after plating and treated with 10 ng/ml staurosporine for
0, 24, and 48 h. 6 h before harvesting, cells were
pulse-labeled with L-[3H]leucine, and the
incorporated L-[3H]leucine was measured with
a scintillation counter. **, p < 0.01.
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Overexpressed CDK11p46 Causes Higher Apoptosis
Rate--
We next addressed whether overexpression of
CDK11p46 increases apoptosis. Vector control or
CDK11p46-overexpressing A375 cells were stained with 7-AAD
and subjected to flow cytometry. Results showed increased apoptosis in
cells stably transfected with CDK11p46 compared with cells
transfected with vector only (Fig.
10A). This observation was
confirmed by cell morphological analysis using light microscopy and
Giemsa staining, which showed that CDK11p46-overexpressing
cells had more apoptotic cells than vector only-transfected cells (Fig.
10B). These results demonstrate that CDK11p46
promotes apoptosis.

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Fig. 10.
CDK11p46 overexpression leads to
increased apoptosis. A, cells stably transfected
with pcDNA3 or pcDNA3-CDK11p46 A375 were
analyzed for apoptosis by 7-AAD staining followed by flow cytometry.
Cells in the region R1 represent normal cells, cells in R2 represent
early apoptotic cells, and cells in R3 represent late apoptotic cells.
The percentages of the cells in R2 and R3 are listed to the
right of the histograms. Left
bars show the percentage of total apoptotic cells. The
figure is a representative result from at least two
independent experiments. B, A375 cells stably transfected
with pcDNA3 vector only or pcDNA3-CDK11p46 were
collected onto slides by cytospin followed by Giemsa staining and light
microscopy analysis. Magnification was ×15. a, A375 cells
stably transfected with pcDNA3 vector only; b, A375
cells stably transfected with CDK11p46.
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DISCUSSION |
CDK11 appears to be downstream of caspase-3 in apoptotic signaling
(2). Upon apoptotic stimulation, CDK11 is cleaved by caspases and
activated (1, 13, 14). However, the potential substrates of CDK11 that
may be involved in cell death are not known. In the present study, we
identify that eIF3 p47 interacts with the caspase-processed CDK11
isoform using a yeast two-hybrid screening strategy.
The eIF3 p47 is a subunit of the mammalian eIF3 multiprotein complex
that binds to the 40 S ribosome and promotes the binding of
methionyl-tRNA and mRNA (21, 25). The eIF3 p47 protein appears to
be a Mov34 family member (26, 27). Mov34 family members are involved in
regulation of proteasome, translational initiation, and transcription
(26). In the present study, we can show that the carboxyl-terminal
domain of CDK11p110 directly interacts with eIF3 p47 in GST
pull-down assays. Furthermore, deletion of DNA sequences of the
Mov34/JAB_MPN domain in eIF3 p47 eliminates the interaction between
these two proteins. Thus, the Mov34/JAB_MPN domain within eIF3 p47 may
be important for the protein-protein interaction with the
carboxyl-terminal domain of CDK11p110.
We also demonstrate by confocal microscopy that both eIF3 p47 and
CDK11p46 co-localize in the nucleocytoplasm. We also show
for the first time that there is both a cytoplasmic and nuclear pool of
eIF3 p47. This phenomenon is not uncommon. Several other proteins, such
as cyclin B1 (28), RanBPM (29), and eukaryotic initiation factors 4E
(eIF4E) (30, 31), eIF4G (32), and eIF5A (33) can also be found in the
nucleus as well as the cytoplasm. Since eIF3 p47 is a Mov34 family
member, it is tempting to speculate that it may have a nuclear function
in mRNA metabolism such as splicing or transport. We are currently
pursuing these possibilities.
In this study, we show that endogenous CDK11p46 and eIF3
p47 protein can interact and that stimulation of apoptosis enhances this interaction. We also show that the kinase activity of CDK11 is
activated during apoptosis, that the kinase obtained can phosphorylate eIF3 p47 apparently at serine 46, and that the phosphorylation of eIF3
p47 by CDK11 can be blocked by caspase-3 inhibitor (DEVD-FMK). In
addition, mutation of the phosphate transfer site in
CDK11p46 abrogates the phosphorylation of eIF3 p47 protein.
Finally, we show that serine phosphorylation of eIF3 p47 occurs in
cells undergoing apoptosis, with maximal phosphorylation occurring at
36 h both in vitro and in vivo. Although not
definitive, these observations strongly suggest that eIF3 p47 may be a
substrate of the caspase-processed CDK11p110 isoform during
apoptosis. We noticed that the protein level of eIF3 p47 also increased
during apoptosis with maximal expression at 36 h after
stimulation. The biological mechanism of this phenomenon is under investigation.
When cells are committed to apoptosis, a remarkable inhibition of the
rate of overall protein synthesis is observed in a variety of cell
types (24, 34, 35). This down-regulation of protein synthesis may
either protect cells against noxious agents and ensure the conservation
of resources needed for survival or activate apoptosis (36). This
inhibition of protein synthesis occurs at the level of polypeptide
chain initiation and is accompanied by the phosphorylation of the
subunit of eIF2 and the caspase-dependent cleavage of
eIF4GI, eIF4GII, eIF4B, p35 subunit of eIF3, minor proportions of the
subunit of eIF2, and the eIF4E-binding protein 4E-BP1 (21, 37). The
rate of protein synthesis is regulated by the phosphorylation status of
several eIFs. eIF4E phosphorylation was reported to strongly decrease
following anti-CD3 and anti-CD4 stimulation in immature DP thymocytes,
which resulted in rapid decreases in protein synthesis and apoptosis
(38). Phosphorylation of eIF2
by double-stranded
RNA-dependent serine/threonine protein kinase was also
correlated with down-regulation of translation initiation and apoptosis
(21, 36, 39, 40). The data from our study suggest that phosphorylation
of eIF3 p47 may also contribute to the alteration of protein synthesis
during apoptosis. The biological consequences of the phosphorylation of
eIF3 p47 during apoptosis are under investigation.
In A375 cells stably expressing either the caspase-processed
carboxyl-terminal domain of CDK11p110 or a phosphate
transfer site mutant CDK11p46M, we show that luciferase
activity and the production of luciferase protein are compromised in
CDK11p46 but not in CDK11p46M cells. In
addition, overexpressed CDK11p46 from stably transfected
cells are capable of interacting with eIF3 in vivo. These
data suggest that the function of the caspase-processed CDK11p110 isoform may be to inhibit translation during
apoptosis. However, whether or not this inhibition of protein
translation occurs in an eIF3 p47-dependent or -independent
manner remains to be clarified.
In summary, the CDK11 protein kinase may be involved in apoptotic
signaling. This study demonstrates that the caspase-processed CDK11p110 isoform can interact with eIF3 p47, which is a
Mov34 family member. The correlation of inhibition of protein synthesis
and eIF3 p47 phosphorylation by CDK11p46 suggests, but does
not prove, a causal relationship between the two phenomena. Further
analysis of this interaction might result in a clearer understanding of
the regulation and function of CDK11 and eIF3 p47, thus providing new
insights into apoptotic signaling and protein synthesis.