CK2 phosphorylation of eukaryotic translation
initiation factor 5 potentiates cell cycle progression
Miwako Kato Homma*†, Ikuo Wada‡, Toshiyuki Suzuki§, Junko Yamaki*, Edwin G. Krebs†¶, and Yoshimi Homma*
Departments of *Biomolecular Sciences and‡Cell Science, CREST, JST, and§Biochemistry, Fukushima Medical University School of Medicine,
Fukushima 960-1295, Japan; and¶Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine,
Seattle, WA 98195-7370
Contributed by Edwin G. Krebs, August 9, 2005
Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser?Thr protein
kinase that plays an important role in cell cycle progression.
Although its function in this process remains unclear, it is known
we show that CK2 activity changes notably during cell cycle
progression and is increased within 3 h of serum stimulation of
quiescent cells. During the time period in which it exhibits high
enzymatic activity, CK2 associates with and phosphorylates a key
molecule for translation initiation, eukaryotic translation initiation
factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are
major sites of phosphorylation by CK2. This is confirmed using eIF5
mutants that lack CK2 sites; the phosphorylation levels of mutant
eIF5 proteins are significantly reduced, relative to WT eIF5, both in
vitro and in vivo. Expression of these mutants reveals that they
have a dominant-negative effect on phosphorylation of endoge-
nous eIF5, and that they perturb synchronous progression of cells
is reduced in these cells, and, in fact, restricted diffusional motion
of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant
lacking CK2 phosphorylation sites, as measured by fluorescence
correlation spectroscopy. These results suggest that CK2 may be
involved in the regulation of cell cycle progression by associating
with and phosphorylating a key molecule for translation initiation.
Disruption of the catalytic subunits (? and ??) is lethal in Saccha-
romyces cerevisiae (5) and disruption of the regulatory ? subunit in
mice leads to early embryonic lethality (6). CK2 phosphorylates a
range of cellular targets in a variety of subcellular sites and appears
to be highly pleiotropic; it is involved in many key biological
functions, including growth and cell cycle control (7), signal trans-
duction (3), circadian rhythms (8, 9), and gene expression (10, 11).
CK2 is also a stress-activated kinase and might participate in the
transduction of survival signals to avoid damage by mutagenic UV
radiation (12, 13). An important role for CK2 in promoting cell
proliferation and transformation has been indicated by several
studies. In mammalian systems, its targeted overexpression in mice
results in the development of T cell lymphoma and mammary
tumorigenesis (5). Despite these findings, there is still much un-
The mechanism by which it is regulated and its precise function in
cell cycle progression and proliferation is still poorly understood.
CK2 activity and stability are believed to be regulated in part by
holoenzyme formation via a self-assembly mechanism and by
phosphorylation. Phosphorylation by p34cdc2of the catalytic ?
subunit at the C-terminal domain occurs in a cell cycle-dependent
manner in mitotic cells. The regulatory ? subunit is also autophos-
phorylated at four sites, including Ser-2, -3, -4, and -209, the latter
being maximally phosphorylated in mitotic cells. So far, no clear
effect of phosphorylation of CK2 on its activity has been demon-
strated. Previously, we described a cell cycle-dependent interaction
between CK2 and the adenomatous polyposis coli (APC) tumor
suppressor protein and an inhibitory effect of APC on CK2 activity
asein kinase 2 (CK2) (1–4) is composed of two subunits, ? or
?? and ?, which combine to form a native ?2?2 tetramer.
with regulatory molecules such as APC rather than by direct
In this work, we demonstrate a significant increase in CK2
activity in cells induced to enter G1 phase by growth factor
stimulation. During this time period, CK2 associates with and
further identify the sites of eIF5 phosphorylation and show that
eIF5 mutants that lack these phosphorylation sites attenuate cell
cycle progression and proliferation. The formation of translation
initiation complexes is also suppressed by the eIF5 mutants, result-
ing in suppression of expression of cell cycle regulators such as
cyclin B1. Our observations suggest that CK2 is involved in regu-
lating translation and the cell cycle through the association and
phosphorylation of eIF5, a key component in translation initiation.
and normal human fetal lung fibroblasts TIG-7 were grown in
DMEM supplemented with 10% FBS. For synchronization exper-
iments, logarithmically growing cells were starved in 0.2% FBS for
48 h and then cultured in fresh media containing 10% FBS for an
additional 16–20 h to obtain cell populations enriched in S phase.
Alternatively, cells were arrested in prometaphase by adding 50
treated with apigenin (Sigma) at 80 ?M for 2 h or with short
interfering RNA (Upstate Biotechnology, Lake Placid, NY) to
inhibit kinase activity.
Plasmids and Transfections. Full-length cDNAs for human CK2?
and -? subunits were obtained as described (16). Human eIF5
cDNA was isolated from a cDNA library of human fetal fibroblast.
and -390 to two Ala residues (M1) (Mn, mutant n of eIF5) and to
and M2. All constructs and mutations were confirmed by DNA
sequencing (for further details, see Supporting Methods, which is
published as supporting information on the PNAS web site).
Kinase Assays. CK2 activity was measured by p81-filter assay by
using RRREEETEEE or RRRDDDSDDD as a substrate peptide
(17). In vitro phosphorylation of eIF5 by CK2?? was assayed by
incubating a reaction mixture consisting of 20 mM Hepes, pH
7.4?10 mM ?-glycerophosphate?5 mM MgCl2?10 ?g/ml aproti-
nin?5 ?g/ml leupeptin?1 mM PMSF?0.2 mM ATP?1 ?Ci [?-32P]
ATP (1 Ci ? 37 GBq), in the presence or absence of 10 ng?ml
heparin at 30°C for 5 to 20 min. Phosphorylation reactions were
Abbreviations: APC, adenomatous polyposis coli; CK2, casein kinase 2; eIF5, eukaryotic
translation initiation factor 5; TIG-7, normal human fetal lung fibroblasts; HEK, human
embryonic kidney; Mn, mutant n of eIF5.
†To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
© 2005 by The National Academy of Sciences of the USA
October 25, 2005 ?
vol. 102 ?
separated by SDS?PAGE (18), and32P incorporation was detected
by autoradiography. For in vivo labeling, see Supporting Methods.
Preparation of whole-cell lysates and the conditions for immuno-
blotting and immunoprecipation for cyclin B1kinase assays have
been described (19).
Mass Spectrometric Analysis. Two eIF5 preparations were analyzed
in HEK293 cells, the cells stimulated with FBS for 6 h after serum
starvation, and the eIF5 protein immunoprecipitated by anti-
FLAG agarose to characterize the phosphorylation state in vivo.
Recombinant eIF5 protein was phosphorylated in vitro by incubat-
separated by SDS?PAGE (12% acrylamide). Bands were excised
and proteins digested with trypsin (Promega). Extracted peptides
were analyzed by using QSTAR electrospray ionization TOF
tandem MS (Applied Biosystems).
Analysis of Protein Complexes by Sucrose Density Centrifugation.
and then treated with cycloheximide for 30 min before harvesting.
Lysates were clarified by centrifugation at 23,000 ? g for 30 min at
between 2 and 4 mg?ml (20). The sample was loaded on a 5–40%
sucrose gradient prepared in a buffer containing 20 mM Tris?HCl,
pH 7.5; 100 mM KCl; and 1 mM MgCl2, and centrifuged at 250,000
? g (SW55Ti, Beckman) for 4.5 h at 4°C. The gradient was
separated into 15 fractions of 0.2 ml, and the absorption of each
fraction at 254 nm was monitored. Each fraction was used for eIF5
Analysis of Protein Dynamics in Living Cells. Introduction of GFP-
fused eIF5 expression plasmids by using siliconized glass mi-
crobeads into cells was described previously (21). For fluorescence
correlation spectroscopy (FCS) analysis, a ConfoCor2 instrument
(Zeiss) was used and analysis carried out as described (22).
Activation of CK2 in Response to FBS Stimuli. To investigate the
control of CK2 activity and expression in relation to cell cycle
progression, we examined changes in its activity in response to
serum stimulation using TIG-7 cells synchronized by serum star-
vation. Expression of cyclin B1was monitored to verify cell syn-
chronization (Fig. 1B). Cell lysates were prepared from unstimu-
FBS for varying time periods. CK2 activity was enhanced in a
time-dependent manner after the stimulation of quiescent cells
when the synthetic peptide RRREEETEEE was used as substrate,
reaching the highest level in the first 3 h and decreasing thereafter
as cells progressed toward the G2?M phase (Fig. 1A). In contrast,
the synthetic peptide RRRDDDSDDD as a substrate. There were
no obvious changes in either CK2? or -? subunit expression during
the time course of the experiment (Fig. 1B), indicating that the
change in kinase activity could be ascribed to specific activity.
for it in cell cycle progression and a possible association with
molecules involved in regulating progression through G1. Thus, we
screened for interacting molecules by analyzing proteins associated
-? were present in the immunoaffinity-purified fraction and were
found to interact with several proteins, including eIF5, by in-gel
digestion and mass spectrometric analysis (Fig. 1C).
Cell Cycle-Dependent Phosphorylation of eIF5. The results described
above raise the possibility that eIF5 is a physiological target for
phosphorylation by CK2. To determine whether eIF5 is actually
phosphorylated in vivo, we prepared extracts from TIG-7 cells
metabolically labeled with32P-orthophosphate and analyzed im-
munoprecipitated eIF5 for radiolabeling. Lysates were prepared
from cells in different cell cycle stages, including serum-starved
cells in G0, cells stimulated with 10% FBS for 3 h in G1, cells
arrested in S phase by double thymidine treatment, and cells
by 3 h after FBS treatment, and increased further in S and G2?M
phases. These changes were confirmed by following cells for 24 h
after serum stimulation (Fig. 2 A, lanes 5–11, and B, lanes 1–4).
Phosphorylation of eIF5 was induced by 3 h after stimulation,
increased further over 10 h, and remained high in cells progressing
through S and G2?M phases. On the other hand, when logarith-
eIF5 phosphorylation level decreased to basal levels over 48 h (Fig.
2C, lanes 5–9). The cell number was constant for up to 72 h during
Next, we examined whether the interaction between eIF5 and
CK2 occurs in a cell cycle-dependent manner. Immunoprecipitates
in the cell cycle and analyzed by immunoblotting using an anti-
CK2? antibody probe. These experiments demonstrated that eIF5
associated with CK2? only in G1, and not in G0or G2?M, phases
(Fig. 2C, lanes 1–4). Time-course experiments also demonstrated
that this association was observable 3 h after FBS stimulation,
increased in a time-dependent manner until 10 h, and quickly
disrupted after 12 h. The highest level of interaction was observed
?8 h after FBS stimulation (Fig. 2C, lanes 5–11).
To determine whether CK2 could be responsible for the phos-
phorylation of eIF5, we tested the ability of this enzyme to
phosphorylate eIF5 in vitro. As shown in Fig. 2D, GST-eIF5 was
readily phosphorylated by recombinant CK2 in vitro, and this
phosphorylation was markedly reduced in the presence of the CK2
inhibitor, heparin, suggesting that CK2 is the kinase responsible for
cells were stimulated with 10% FBS and collected at the indicated times after
treatment. (A) CK2 activity recovered in the immunoprecipitates with anti-CK2?
antibody was measured by using two synthetic peptide substrates. Specific ac-
tivity was calculated by subtracting radioactivity in the absence of peptide sub-
antibodies. (C) Protein profile of immunoprecipitates with anti-CK2? antibody
from TIG cells stimulated with FBS for 3 h. Proteins were subjected to SDS?PAGE
and visualized by Coomasie brilliant blue (CBB) staining.
Activation of CK2 by growth-promoting stimuli. Serum-starved TIG-7
Homma et al. PNAS ?
October 25, 2005 ?
vol. 102 ?
no. 43 ?
phosphorylation of eIF5. Similar results were obtained when the
recombinant CK2 was substituted with endogenous enzyme that
was purified from logarithmically growing TIG-7 cells (Fig. 2D,
lanes 7–11). Based on these results, we predicted that inhibition of
CK2 activity would reduce phosphorylation of eIF5 in vivo. TIG-7
cells were treated with the selective CK2 inhibitor apigenin or were
pretreated with short interfering RNA to suppress the expression
of CK2 and then labeled with32P-orthophosphate after serum
stimulation. As shown in Fig. 2E, in vivo phosphorylation of eIF5
was attenuated as expected. Thus, both pharmacologic and molec-
ular inhibition of CK2 demonstrates that eIF5 is phosphorylated
by peptide mass analysis and peptide sequencing (tandem MS).
Recombinant eIF5 protein was phosphorylated with human
CK2?? holoenzyme for 20 min, then digested with trypsin and
analyzed by QSTAR-TOF MS. We compared the peptides from
that increase in mass by 80 Da. As a result, two phosphorylated
serine residues, Ser-389 and -390, were unambiguously identified
(Fig. 3A). The mass of a fragment ion from the phosphorylated
peptide was 160 Da larger than that of the unphosphorylated
(Fig. 3B). Similar spectra of the phosphoforms of the peptide were
observed when in vivo-labeled eIF5 was digested for mass spectro-
metric analysis. Phosphorylation of Thr-207 and -208 in eIF5 was
also detected, although these sites were phosphorylated to a lesser
extent than the Ser sites.
Inhibition of eIF5 Phosphorylation by Mutation of CK2 Sites. To
confirm these phosphorylation sites, a series of site-directed mu-
tants were engineered and confirmed by sequencing. We con-
structed plasmids for expression of FLAG-tagged WT eIF5,
FLAG-tagged eIF5 in which Ser-389 and -390 were substituted by
Ala (Ser-389?390-Ala, M1), FLAG-tagged eIF5 in which Thr-207
and -208 were substituted by Ala (Thr-207?208-Ala, M2), and
FLAG-tagged eIF5 possessing all four mutations (Ser-389?390-
Ala–Thr-207?208-Ala, M3) as shown in Fig. 3A. Although the
FLAG-tagged WT eIF5 was phosphorylated after 6 h of serum
stimulation, only a small proportion of the mutant proteins were
phosphorylated under similar expression conditions (Fig. 3C, lanes
3–5). Mutants M1 and M3 exhibited much lower levels of phos-
phorylation than the WT in vivo. It is noteworthy that phosphor-
ylation of the endogenous eIF5 protein was suppressed by the
expression of eIF5 mutant proteins (Fig. 3C, lanes 8–10): in
particular, M1 almost completely suppressed phosphorylation of
endogenous eIF5, and M2 and M3 had similar effects, although to
a lesser extent. These results indicate a dominant-negative effect of
the eIF5 mutants on phosphorylation levels of endogenous eIF5
protein and indicate the importance of phosphorylation at Ser-389
and -390 on eIF5.
The ? Subunit of CK2 Is Necessary for eIF5 Phosphorylation. As to
presence of the regulatory ? subunit. The CK2? subunit plays an
important role in the assembly of tetrameric CK2 complexes, in
enhancing the catalytic activity and stability of CK2, and in mod-
that CK2? is responsible for the docking and?or recruiting of CK2
substrates or potential regulators. In this respect, potential CK2
targets as well as potential CK2 regulators interact with CK2 via
interactions with CK2?.
by CK2? in vitro and whether CK2 is the only kinase that phos-
eIF5 and CK2? were incubated for 10 min in the presence of
different concentrations of CK2? or of cyclin?CDK6 kinase, PKA,
or mitogen-activated protein kinase (MAPK) instead of CK2?. As
shown in Fig. 4A, phosphorylation of eIF5 increased as the molar
phosphorylated eIF5 under the conditions where CK2? phosphor-
ylates the substrate in the presence of the CK2? subunit. Recom-
binant mutants of eIF5 in which Ser-389 and -390 were replaced by
alanine were also resistant to phosphorylation by CK2 in the
depended on the concentration of CK2? (Fig. 4B). These results
suggest that CK2 is the sole kinase acting on eIF5, phosphorylating
mainly Ser-389 and -390.
Phosphorytion of eIF5 for Translation Initiation Assembly. eIF5 is
involved in the formation of part of a multifactor complex com-
sion. (A) Serum-starved TIG-7 cells were stimulated with
10% FBS for varying time periods and labeled with32P-
orthophosphate for the final 1 h. Cell lysates were pre-
and radioactive bands were visualized by autoradiogra-
phy. (Left) G0, unstimulated cells (lane 1); G1, stimulated
stimulated with 10% FBS for the various periods indi-
cated (lanes 6–11). (B) Phosphorylation levels of eIF5
were associated with growing state. Cell lysates were
prepared as in A (lanes 1–4), growing nonsynchronized
cells (lane 5), and cells starved by lowering the serum
concentration to 0.2% for 6–48 h (lanes 6–9). (C) Asso-
ciation of eIF5 and CK2?. Immunoprecipitates obtained
from cells as in A with an anti-eIF5 antibody were sepa-
rated by SDS?PAGE and blotted with anti-CK2? anti-
body. (D) In vitro phosphorylation of eIF5 by CK2. Phos-
phorylation mixtures containing recombinant eIF5 and
CK2?? proteins were incubated at 30°C, as indicated, in
the absence (lanes 1–5) or presence (lane 6) of 10 ng?ml
heparin. The CK2?? holoenzyme recovered in immuno-
precipitates from growing TIG-7 cells was used in lanes
jected to autoradiography. (E) Inhibition of CK2 reduced phosphorylated eIF5 levels. Serum-starved TIG-7 cells were stimulated for 6 h and treated with apigenin
(CK2I?), left untreated (?), pretreated with CK2–short interfering RNA (siRNA?) or left untreated (?), then starved and stimulated for 6 h. eIF5 protein was labeled
and immunoprecipitated as in A. Total lysates were analyzed in parallel for eIF5 protein content (Lower).
www.pnas.org?cgi?doi?10.1073?pnas.0506791102 Homma et al.
eIF4 (23, 24). We examined the effect of the CK2-mediated
phosphorylation of eIF5 on the formation of this translation–
initiation complex. Sucrose gradient-velocity sedimentation was
used to fractionate lysates from cells expressing WT or M1 eIF5,
examined. As shown in Fig. 5 A and B, the expression of M1 eIF5
shifted the distribution of each component of the translation–
cells, the majority of eIF2 and eIF3 was detected in fractions 4 or
5, respectively, coeluting with the ribosomal S6 protein peak, and
eIF5 was mainly detected in fractions 5–10. In contrast, eIF3 was
recovered in fractions 4 and 5 of M1-expressing cells, and only a
trace amount of eIF2 was found in fraction 6, which was behind the
peak of eIF3. eIF5 was observed in fractions 7–10 and in lighter
fractions (data not shown).
To confirm that reduced phosphorylation of eIF5 disrupts com-
plex formation, eIF5 WT and M1 mutant were probed for inter-
actions with eIF2. FLAG-tagged eIF5 WT and M1 DNA were
independently expressed in HEK293 cells, which were then ana-
lyzed in an attempt to identify interactions of these proteins with
disrupted the interaction between WT eIF5 and eIF2 in vitro (Fig.
5C Lower). These results suggest that the phosphorylation of eIF5
by CK2 is important for the proper association of eIF5 with the
mature translation–initiation complex, at least with eIF2, which is
engaged in translation in vivo.
FCS Measurements of eIF5. Next, we analyzed how the M1 mutation
affects the association of eIF5 with the translational machinery by
using FCS. This method measures fluctuation of fluorescent mol-
ecules in a subfemtoliter confocal volume at very high time reso-
lution (25). By calculating the autocorrelation function of such
fluctuating fluorescence signals, complex formation with relatively
immobile molecules can be detected in living cells. Because eIF5
forms a large complex with other members of eIF as well as
ribosomes, and the current results predict that CK2 phosphoryla-
tion of eIF5 is required for functional complex formation, we
from that of the WT molecule.
In Fig. 5D, our measurements of both the GFP-tagged eIF5 WT
indicates that the M1 mutant diffused in a less-restricted manner
than the WT. Fitting of the WT eIF5 diffusion profile to the
of two types of slow components other than the fast simple
diffusional component; one is in the order of milliseconds and the
as supporting information on the PNAS web site). It is likely that
the millisecond diffusion fraction represents the molecules associ-
ated with other eIFs or translation complexes tethered to mem-
branes, because diffusion in this range is typical of membrane
proteins. In the M1 autocorrelation curve, the slowest fraction was
not detected, and the millisecond diffusion was markedly reduced.
Consistent with this, GFP-eIF5 WT showed large deviation from
the one-compartment model, whereas the M1 curve moderately
resembled the one-component model, although the two-
compartment model was best-fitted (Fig. 5E). This indicates that
GFP-eIF5 WT requires CK2 phosphorylation sites to interact with
the relatively immobile molecular machinery such as the putative
initiation complexes on ribosomes.
peptide sequence corresponds to residues 384-EAEEESSGGEEEDEDENIEVVY-405 of human eIF5. Both Ser-389 and -390 of eIF5 are phosphorylated by CK2. (C)
Ala mutations at phosphorylation sites in eIF5 were engineered, and those phosphorylation levels were monitored in vivo. COS-7 cells expressing WT or mutant
of FLAG-tagged eIF5 were stimulated for 6 h, then labeled as shown in Fig. 2. Mock transfected (lanes 1 and 6), WT (lanes 2 and 7), M1 (lanes 3 and 8), M2 (lanes
4 and 9), and M3 (lanes 5 and 10) were used for immunoprecipitation with anti-FLAG (lanes 1–5) or -eIF5 (lanes 6–10) antibodies. (Lower) The amount of
immunoprecipitated eIF5 was determined by immunoblotting with anti-eIF5 antibodies.
Homma et al. PNAS ?
October 25, 2005 ?
vol. 102 ?
no. 43 ?
above results suggest that eIF5 mutants may affect translation
initiation and cell proliferation. Therefore, we monitored COS-7
cells expressing FLAG-eIF5 mutants by using MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays.
Cell proliferation was significantly abrogated by all three mutants
and reached 30% reduction in M1- compared with WT eIF5-
expressing cells. In contrast, when using eIF5 mutants in which the
glutamic acids, cell proliferation was enhanced by 30–50% in M4-,
M5-, or M6-expressing cells that mimic phosphorylated states in
eIF5 (Fig. 7A, which is published as supporting information on the
PNAS web site). DNA histogram analysis was carried out to
quantify populations in G0?G1, S, and G2?M stages, and the
populations in the G2?M phase and an increase in the G0?G1and
S phases (Fig. 7B).
Because cyclin B1is an important regulator of G2?M transition
(26), we determined its expression level in HEK293 cells that were
transfected with WT or M1 eIF5. For reference, we also analyzed
cells that were transfected with the CK2?-subunit or APC protein
C-terminal fragment (APC-C) that is a negative regulator of CK2
activity (15). M1 expression reduced cyclin B1 levels by ?40%.
and CK2? had no effect on the content of cyclin B1. Also, cyclin
B1-asociated kinase activity was significantly inactivated by M1 or
APC-C expression (Fig. 8, which is published as supporting infor-
mation on the PNAS web site). This verifies that blocking phos-
phorylation of eIF5 by site-directed mutagenesis inhibits cell cycle
in a variety of cell types, definitive evidence that this kinase is
regulated in response to growth factor stimuli has been difficult to
reproduce (14). In this study, we successfully demonstrated a
dramatic and reproducible enhancement of CK2 activity in re-
was activated 5-fold by 3 h after stimulation and returned to basal
levels 7 h later. Because CK2 has been considered to be constitu-
tively active and not regulated by second messengers, mechanisms
for enhancement of its activity in cells includes gene expression,
covalent modifications such as phosphorylation, and interactions
with other cellular molecules. The results presented in Fig. 1 show
no change in CK2? or -? levels over the 24 h after FBS stimulation.
phosphorylation in immunoprecipitates with eIF5. However, in-
Analysis of eIF5 phosphorylation by CK2 in vitro. Phosphorylation mixtures
containing recombinant eIF5 were incubated at 30°C for 10 min in the pres-
ence of CK2? at molar ratio of ??? of 0, 0.1, 0.2, 0.5, 1.0, respectively, or in the
presence of cyclin?CDK6 kinase, PKA, or mitogen-activated protein kinase,
and then separated by SDS?PAGE and autoradiographed as in Fig. 2. (B)
Mutation of Ser-389 and -390 to alanine in eIF5 reduced phosphorylation by
CK2. The molar ratio of CK2? to ? is 0.5 (?) and 1.0 (??), respectively.
Recombinant eIF5 protein in the phosphorylation reactions is shown (Lower).
The ? subunit of CK2 is necessary for eIF5 phosphorylation. (A)
mutant eIF5-expressing cells. Cell lysates were prepared from exponentially
growing cultures of HEK293 cells transfected with either WT (A) or M1 (B)
FLAG-eIF5, and each sample (?2 mg of protein) was separated on a 5–40%
(wt?vol) sucrose gradient. Absorbance at 254 nm was measured. Anti-FLAG
agarose beads were added to each eluate to separate the FLAG protein, then
to estimate the 40S–43S ribosome fractions. Representatives of five separate
experiments are shown. (C) eIF5 mutant disrupt interaction with eIF2. (Upper)
FLAG-eIF5 WT or M1 was expressed in HEK293 cells and immunoprecipitated by
using anti-FLAG agarose resin. (Lower) GST-eIF5 WT or M1 proteins were incu-
bated separately with HEK293 cell lysates for 4 h at 4°C and pulled down by
(D) FCS measurements of GFP-eIF WT and M1. COS7 cells were transfected with
pEGFP-eIF5 and incubated for 3 h at 37°C. Sixty-five spots in the cytoplasm were
Of these spots, 47 (WT) or 60 (M1) were used for calculating the average auto-
correlation function. The normalized autocorrelation is plotted for GFP-eIF5 WT
(red) and M1 (blue). The diffusion time and fraction of each component were
estimated by fitting to three- (WT) or two-component (M1) models.
Analysis of the translation initiation protein complex profile of WT and
www.pnas.org?cgi?doi?10.1073?pnas.0506791102Homma et al.
creased recovery of CK2? in immunoprecipitates with eIF5 was Download full-text
imply that the activity of CK2 is controlled by interaction with
regulatory molecules rather than by protein expression or direct
phosphorylation. In this context, our previous results demonstrat-
(15), although the explicit mechanism of the molecular interaction
is still unclear.
In this study, we demonstrate that CK2 interacts with and
phosphorylates eIF5, a key molecule for the formation of the
translation initiation complex. The association is observed when
CK2 activity is elevated by growth factor stimulation. Phosphory-
lation sites in eIF5 catalyzed by CK2 were identified, and mutants
lacking these sites were verified to eliminate phosphorylation by
CK2. We demonstrated that phosphorylation of Thr-207 and -208
of acidic residues on both sides of Thr-207 and -208 may evoke the
possible involvement of phosphorylation at these sites for efficient
phosphorylation at Ser-389 and -390. Furthermore, these mutants
exhibited dominant-negative effects on phosphorylation of endog-
enous eIF5. Based on these results, we conclude that eIF5 is an
intracellular target for phosphorylation by CK2.
release of eIF2-GDP and eIF3 from the 40S subunit, which is
essential for the subsequent joining of the 60S ribosomal subunit
with the 40S complex to form an elongation-competent 80S initi-
ation complex. Our data show that the transition from the 40S to
of the M1 mutant, indicating an important role for CK2-mediated
phosphorylation of eIF5 on the maturation of the complex. Based
on these results, we hypothesize that CK2-mediated phosphoryla-
as a GTPase-activating protein for eIF2. A role for eIF5 in the
maturation of the complex has been supported by findings in yeast.
the C terminus of eIF5 to modulate close interaction and deletion
of 7–12 amino acids from the AA-boxes, leads to destruction of the
multifactor complex, and diminishes the fraction of ribosomes
engaged in translation in vivo (20, 27, 28). Because the CK2-
mediated phosphorylation sites Ser-389 and -390 are located in the
AA-box (Fig. 3A), it is reasonable to suggest that phosphorylation
within the AA-box regulates interaction with other initiation
indicate that the WT protein was immobilized by associating with
a large protein complex. In contrast, random diffusion of the
GFP-eIF5 mutant in a real-time manner revealed significant pro-
longation, compared with that of the WT. This indicates that
GFP-eIF5 WT has a significant interaction with the putative
translation complex through CK2 phosphorylation sites, thus ran-
dom diffusion of the eIF5 mutant is increased when the latter lacks
The phosphorylation level of eIF5 was found to be quite low in
stimuli. This high level was maintained during progression of the
two molecules were dissociated. Eliminating growth factors from
the culture medium was sufficient to reduce the phosphorylation
It is noteworthy that CK2-mediated phosphorylation of eIF5
might be responsible for cell cycle progression. We demonstrate
that removal of CK2 phosphorylation sites in eIF5 attenuates cell
cycle progression and causes reduced S to M transition. In partic-
ular, expression of M1 leads to an obvious reduction in the cell
population in M phase and increases the population in G1and S
phases. These results indicate a mechanism in which eIF5 phos-
phorylation by CK2 is needed for normal cell cycle progression at
the S?G2boundary. Indeed, the expression level of cyclin B1, and
eventually its associated kinase activity that are essential for entry
into mitosis, was reduced by M1 eIF5 mutant expression (Fig. 8). A
APC protein C-terminal fragment protein, an endogenous CK2
inhibitor. Therefore, we conclude that CK2-dependent phosphor-
ylation of eIF5 is required to assemble the normal translation
initiation complex, which may stimulate the translation of cell cycle
needed for mitotic entry. Notably, this effect is specific, because no
significant changes in expression were observed for cyclin A, D, or
E (data not shown). In this context, eIF5 phosphorylation by CK2
might be an important modifier for the progression of cell cycle
from S to G2?M phase by reflecting translation status.
Collectively, these data suggest that CK2 may participate in the
eIF5 phosphorylation constitutes an important molecular event for
the progression of the cell cycle.
We thank Drs. Dongxia Li, Natalie G. Ahn, and Tamiko Kano-Sueoka
for discussions on the manuscript. This work was supported by grants
from the Ministry of Education, Science, Sports and Culture of Japan,
and from the Fukushima Society for the Promotion of Medicine.
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vol. 102 ?
no. 43 ?