JOURNAL OF VIROLOGY, Apr. 2004, p. 3502–3513
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 7
High-Throughput Screening of the Yeast Kinome: Identification of
Human Serine/Threonine Protein Kinases That Phosphorylate the
Hepatitis C Virus NS5A Protein
Carlos Coito,1Deborah L. Diamond,1Petra Neddermann,2Marcus J. Korth,1
and Michael G. Katze1,3*
Department of Microbiology1and Washington National Primate Research Center,3University of Washington,
Seattle, Washington 98195, and IRBM Merck, Rome, Italy2
Received 7 October 2003/Accepted 4 December 2003
The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance
to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphopro-
tein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report,
we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged
protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable
of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the
sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest
sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that
several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopep-
tide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and
MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin,
an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of
specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this
group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA
translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The
findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the
major task of identifying human NS5A protein kinases for further characterization of phosphorylation events
in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other
protein biochemical activities by mechanistic class.
Phosphorylation is a common posttranslational processing
event in the synthesis of proteins, and changes in the degree of
protein phosphorylation play an important role in modulating
protein function. Many cellular processes, including signal
transduction, transcription, protein synthesis, and cell growth
and differentiation, are regulated by phosphorylation-depen-
dent mechanisms (17, 19). Therefore, it is not surprising that
the protein kinases, a family of enzymes that catalyze the
phosphorylation of proteins, comprise one of the largest fam-
ilies of genes in eukaryotes. The protein kinase complement of
the human genome (the human “kinome”) is estimated to
comprise about 1.7% of all human genes, with a total of 518
genes identified according to a cataloging approach published
recently (19). The presence of such a large number of kinases
in the human genome represents a significant challenge to
protein kinase studies, suggesting that it would be extremely
powerful to develop high-throughput approaches for charac-
terizing kinase-substrate reactions. Although a recent study
describes the use of a microarray format for the high-through-
put analysis of substrate specificity of yeast protein kinases
(36), the use of such an approach to identify the human ki-
nase(s) responsible for phosphorylation of a biological sub-
strate has not been previously described.
There has been much interest in characterizing the role of
protein phosphorylation in mechanisms associated with viral
infection, pathogenesis, and persistence (16, 35). In this regard,
it is well established that the hepatitis C virus (HCV) nonstruc-
tural 5A (NS5A) protein is a phosphoprotein and that phos-
phorylation of NS5A is highly conserved among HCV geno-
types and other members of the Flaviviridae family (13, 23, 25).
This led to the idea that phosphorylation may play a role in the
flavivirus life cycle and inspired our interest in obtaining a
better understanding of NS5A phosphorylation, particularly
since this protein has been implicated in resistance of HCV to
interferon treatment (4, 5, 27). NS5A is phosphorylated pri-
marily on serine residues both in vitro and in vivo (13, 15, 22,
23, 25, 30). Although the effects of various kinase inhibitors
have implicated a cellular serine/threonine kinase(s) from the
CMGC kinase group (25), it should be noted that this group
contains several kinase families, and these studies did not iden-
tify a particular kinase in the phosphorylation of NS5A. More
recent in vitro kinase assays specifically demonstrated that
NS5A can be phosphorylated by casein kinase 2 (CK2) (12, 14)
and protein kinase A (PKA) (12).
The interferon-induced double-stranded RNA-activated
protein kinase, PKR, is a key component of the antiviral and
antiproliferative effects induced by interferon. NS5A forms a
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Washington, Box 358070, Seattle, WA 98195-8070.
Phone: (206) 732-6135. Fax: (206) 732-6056. E-mail: honey@u
complex with PKR, disrupting PKR dimerization, which results
in repression of PKR function and inhibition of PKR-mediated
eIF-2? phosphorylation. Whether NS5A is a substrate of PKR
is yet to be determined. There is no PKR homologue in the
yeast kinome, but the yeast kinase GCN2 still has sequence
homology with PKR. In this study, we were particularly inter-
ested in finding out whether GCN2 is a kinase of NS5A.
In this report, we further investigated the phosphorylation of
NS5A using a kinome-scale high-throughput screening ap-
proach in order to identify human protein kinases that phos-
phorylate NS5A. The approach is based on a biochemical
genomics strategy, originally described by Martzen et al. (20),
for constructing an array of molecular constructs for expres-
sion of all yeast kinases (the yeast kinome) (11, 19). Our
long-term interests are to gain insight into the role of phos-
phorylation in the ability of NS5A to modulate a multitude of
cellular signaling pathways. Although there are already more
than 518 known human kinases, more than four times the
number of the kinases in yeast, we reasoned that the yeast
kinome would represent a good model for beginning a global
inspection of NS5A phosphorylation activity, since phosphor-
ylation of NS5A occurs primarily at serine residues (13, 15, 22,
23, 25, 30) and the major families of serine/threonine kinases
are conserved between yeast and humans. The use of the yeast
model was based on the idea that the kinase(s) phosphorylat-
ing NS5A is very likely to be evolutionarily conserved. This is
supported by our previous findings that Ser 2194 is a major
phosphorylation site among all HCV genotypes and this site
can be phosphorylated by yeast, insect, and mammalian ki-
nases (13). We screened 119 glutathione S-transferase (GST)-
tagged protein kinases purified from Saccharomyces cerevisiae
in a global effort to identify yeast kinases capable of phosphor-
ylating NS5A in vitro. BLAST searches were performed to
identify the closest human sequence homologs, and commer-
cially available sources of human protein kinases were used for
subsequent in vitro NS5A kinase assays. We demonstrated that
several of these human kinases were capable of phosphorylat-
ing NS5A in vitro, some of which play a major role in protein
translation and antiapoptotic pathways that are regulated by
NS5A. Furthermore, the phosphopeptide pattern of NS5A iso-
lated from COS-1 cells suggests that the phosphorylation sites
used in vivo were similar to those observed in vitro, allowing us
to identify kinases of particular interest for further character-
ization of NS5A phosphorylation. Finally, rapamycin studies
revealed that p70S6 kinase and potentially closely related fam-
ily members phosphorylate NS5A inside the cell.
MATERIALS AND METHODS
Reagents. The human kinases AKT1, MEK1, MKK6, MKK7?1, SGK1,
p70S6K, CHK2, PDK1, and the rat kinases CK1 delta and p90S6K were pur-
chased from Upstate Biotechnology (Lake Placid, N.Y.). The purity and activity
of these kinases were certified by the manufacturer but were not independently
verified in this study. All materials used for GST-yeast kinase purification were
obtained from Amersham Biosciences Corp. (Piscataway, N.Y.). Radiolabeled
ATP, [?-32P]ATP, and [32P]orthophosphoric acid in water were purchased from
NEN Life Science Products, Inc. (Boston, Mass.). Dulbecco’s modified eagle
medium (DME), phosphate-free DME, Hanks’ balanced salt solution, and calf
serum were purchased from Gibco/Invitrogen Corp. (Grand Island, N.Y.). The S.
cerevisiae protein kinases (119 constructs), cloned in the plasmid pYEX4T-1 and
expressed in the EJ758 yeast strain (36), were provided by Eric M. Phizicky
(University of Rochester, Rochester, N.Y.).
Purification of GST-yeast kinases. Colonies representing transformants of the
EJ758 yeast strain were obtained on plates containing synthetic minimal (SD)
minus Ura media. These colonies were then used to inoculate SD minus Ura
liquid medium and grown for 8 h. At this time, each inoculum was split 1 to 10
into SD minus Ura minus Leu medium and grown for an additional 16 h. When
the cultures reached an optical density at 600 nm of 0.8, the cells were induced
with 0.5 mM CuSO4for 2 to 3 h prior to harvesting by centrifugation at 4°C for
5 min at 1,500 ? g. After induction, the cells were washed twice in two to four
pellet volumes of ice-cold water. The washed cell pellet was resuspended in 1 ml
of homogenization buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 4 mM
MgCl2, 1 mM dithiothreitol [DTT], 10% glycerol, 250 mM NaCl) containing 1?
protease inhibitor mix, and extracts were made with glass beads (15 pulses, 30 s
each at 4°C) using a multichannel vortexer. Extracts were transferred to 15-ml
conical tubes and centrifuged at 4°C for 5 min at 450 ? g. The supernatants were
then centrifuged at 4°C for 30 min at 10,000 ? g. The GST-kinases were purified
from the resulting supernatants by glutathione-agarose chromatography (20).
Briefly, the supernatants were incubated with prewashed reduced glutathione-
agarose beads for 1 h at 4°C and then extensively washed in the same buffer (50
mM Tris-HCl [pH 7.5], 500 mM NaCl, 10% glycerol, 4 mM MgCl2). Aliquots of
33% slurries (150 ?l each) were flash frozen in an ethanol-dry ice bath and stored
at ?80°C for later use.
In vitro yeast kinase assay. Baculovirus recombinant NS5A (2 ?l, 100 ng),
purified from Sf9 cells as previously described (13), was added to 50 ?l of purified
GST-kinase bound to glutathione-agarose beads prepared as described above.
The final amount of kinase added was normalized based on the relative recovery
of each purified kinase as determined by Western blotting with an antibody to
GST. The phosphorylation reaction was carried out at 30°C for 60 min in a final
volume of 60 ?l containing yeast kinase buffer (50 mM Tris-HCl [pH 7.2], 10 mM
MgCl2, 1 mM DTT) supplemented with protease and phosphatase inhibitors and
a mixture of [?-32P]ATP and ATP with a predetermined specific activity (?5,000
dpm/pmol of ATP), such that the reaction mixture contained at least a 1,000-fold
excess of ATP over the NS5A protein concentration. Human recombinant casein
kinase 2 (hCK2) was used as a positive control to demonstrate NS5A phosphor-
ylation. Glutathione-agarose beads, incubated with the yeast EJ758 strain ex-
pressing an empty glutathione vector, were used as a negative control to dem-
onstrate that phosphorylation of NS5A resulted from the appropriate GST-
kinase and not from the nonspecific binding of other proteins to either GST or
the beads. The kinase reaction was terminated by flash freezing the samples in an
ethanol-dry ice bath. An equal volume of 2? SDS protein loading buffer (125
mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 2% ?-mercaptoethanol, 0.02%
bromophenol blue) was added, and the samples were boiled at 95 to 100°C for
5 min prior to analysis by SDS-polyacrylamide gel electrophoresis (PAGE) (10%
gel). After resolving the samples, the gel was dried using a slab gel dryer SGD
4050 (Savant) for 5 h at 80°C and then exposed to X-ray film at ?70°C with an
In vitro human kinase assay. The phosphorylation reaction was carried out in
the presence or absence of 100 ng of baculovirus recombinant NS5A (purified
from Sf9 cells) in a final volume of 40 ?l of human kinase buffer (20 mM
morpholinepropanesulfonic acid [pH 7.2], 25 mM ?-glycerol phosphate, 5 mM
EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 10 mM MgCl2) containing a
mixture of [?-32P]ATP and ATP with a predetermined specific activity (?5,000
dpm/pmol of ATP) such that the reaction mixture contained at least a 1,000-fold
excess of ATP over the NS5A protein concentration. The appropriate kinase was
added and assayed for phosphorylation of NS5A. The buffer condition used was
that recommended by the enzyme supplier. The kinases assayed, and the amount
used per reaction, were 20 mU of rat casein kinase 1 (rCK1 delta), 0.5 ?g of
hAKT1, 0.5 U of hMEK1, 0.05 ?g of hMKK6, 0.2 ?g of hMKK7?1, 0.025 ?g of
hSGK1, 10 mU of hp70S6K, 20 mU of rp90S6K, 10 ng of hPDK1, 100 mU of
hCHK2, or 0.05 ?g of hCK2. For samples containing rCK1 delta, hAKT1,
hSGK1, hCHK2, hp70S6K, or hCK2 (positive control), the reaction was allowed
to proceed for 10 min at 30°C. For samples containing hPDK1, rp90S6K,
hMEK1, hMKK6, hMKK7?1, or hCK2 (positive control), the reaction was al-
lowed to proceed for 30 min at 30°C.
Two-dimensional phosphopeptide mapping. For two-dimensional phos-
phopeptide mapping, the phosphorylated samples were resolved by SDS–10%
PAGE and then transferred to a nitrocellulose membrane. Radiolabeled phos-
phoproteins were detected by autoradiography. The32P-containing bands were
excised, kept wet in water, and incubated in 0.5% polyvinylpyrrolidone–100 mM
acetic acid for 1 h at 37°C in order to block nonspecific adsorption of proteases.
Control slices were prepared from the same-molecular-weight region of lanes
corresponding to phosphorylation reactions containing kinase only or NS5A
only. The slices were then washed thoroughly with deionized water (five times, 1
ml per wash) followed by 50 mM ammonium bicarbonate, pH 8.3 (five times, 1
VOL. 78, 2004SCREEN FOR KINASES THAT PHOSPHORYLATE NS5A 3503
ml per wash), and finally resuspended in 80 ?l of 50 mM ammonium bicarbonate,
pH 8.3. Proteolytic digestion was accomplished by adding 1 ?g of trypsin and
incubating for 3 h at 37°C, followed by another 1-?g addition and incubation at
37°C overnight. The next day, a third addition of 1 ?g of trypsin was made, and
the samples were incubated for 2 h at 37°C prior to adding 1 ?g of chymotrypsin
and digesting for 3 h at 37°C. A second addition of 1 ?g of chymotrypsin was
made, and the samples were incubated at 37°C for 3 h or overnight. The super-
natants containing soluble peptides were then transferred to a separate tube, and
the slices were washed with 400 ?l of water to extract any remaining peptides.
The pooled supernatant and wash were dried with a speed vac, and the peptides
were subsequently suspended in 10 ?l of buffer (7.8% acetic acid, 2.2% formic
acid). Phosphopeptide recovery was monitored at every step by Cerenkov count-
ing. The resuspended samples were loaded onto cellulose thin-layer chromatog-
raphy (TLC) plates (20 cm by 10 cm) and electrophoresed in the first dimension
(20 cm) at 1,000 V for 60 min in reverse polarity (positive to negative). After the
plates were dried overnight in the fume hood, separation in the second dimen-
sion was achieved by ascending chromatography in 37.5% n-butanol, 25% pyri-
dine, and 7.5% acetic acid. The plates were dried extensively, and radiolabeled
phosphopeptides were detected by autoradiography.
In vivo [32P]orthophosphoric acid labeling. COS-1 cells (0.5 ? 106) were
cultured in a T-25 flask for 15 h prior to transfection with 10 ?g of a pcDNA3
construct encoding the NS5A-1b protein. Transfections were performed using
the SuperFect kit (Qiagen, Studio City, Calif.) as described by the manufacturer.
A second flask was transfected with 10 ?g of empty pcDNA3 vector for use as a
negative control. After 15 h of gene expression, the cells were washed with
phosphate-free DME prior to labeling. The cells were then labeled by incubating
each T-25 flask with 1.5 ml of phosphate-free DME supplemented with 10%
dialyzed calf serum (Gibco/Invitrogen Corp.) and [32P]orthophosphoric acid in
water (1 mCi/T-25 flask) for 4 h at 37°C. Each flask was then washed twice with
1 ml of Hanks’ balanced salt solution (Gibco/Invitrogen Corp.) prior to incuba-
tion in 80 ?l of Triton lysis buffer (20 mM Tris-HCl [pH 7.5], 75 mM NaCl, 75
mM KCl, 20% glycerol, 4 mM MgCl2, 1 mM DTT, 1 mM EDTA, 0.5% TX-100)
containing 1? protease inhibitor mix and phosphatase inhibitors (1 mM
Na3VO4, 25 mM ?-glycerophosphate) for 15 min at 4°C. The cells were then
removed from the flasks with a cell scraper, and insoluble materials were pelleted
by centrifugation at 16,000 ? g for 1 min. The resulting supernatants were used
for immunoprecipitation of NS5A as described below.
Inhibition of the mTOR/p70S6K pathway using rapamycin. COS-1 cells (0.5 ?
106, in a T-25 flask) were transfected as described under “In vivo [32P]orthophos-
phoric acid labeling.” The cells were washed with phosphate-free DME and then
incubated with or without 20 nM rapamycin. After 1 h at 37°C, the cells were
labeled with phosphate-free DME supplemented with 10% dialyzed calf serum
(Gibco/Invitrogen Corp.) and [32P]orthophosphoric acid in water for 4 h. The
cells were then processed as described under “In vivo [32P]orthophosphoric acid
Immunoprecipitation of NS5A from cell lysates. Protein lysates prepared from
0.5 ? 106cells were incubated with 1 ?g of anti-NS5A monoclonal antibody for
3 h at 4°C and then mixed with 50 ?l of protein G-agarose beads (Boehringer
Mannheim) for 2 h at 4°C (final volume, 1 ml). The beads were then washed
three times with Triton lysis buffer containing 1? protease inhibitor mix and
phosphatase inhibitors (1 mM Na3VO4, 25 mM ?-glycerophosphate) prior to
suspension in 50 ?l of 2? SDS protein loading buffer (20% [vol/vol] glycerol, 4%
[wt/vol] SDS, 0.125 M DTT). The immunoprecipitated NS5A was then resolved
by SDS–10% PAGE and transferred to a nitrocellulose membrane, and the
NS5A phosphorylation band was detected by autoradiography.
Immunoblot analysis of NS5A expressed in COS-1 cells. Immunoblot analysis
was performed as previously described (28). Briefly, blots were probed with a
monoclonal antibody against NS5A (4 ?g/ml; ViroStat, Portland, Maine), fol-
lowed by a 1:20,000 dilution of donkey anti-mouse immunoglobulin G conju-
gated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, Pa.). NS5A was then visualized by enhanced chemiluminescence
(Amersham Biosciences Corp.).
RESULTS AND DISCUSSION
In vitro phosphorylation of NS5A by yeast protein kinases.
Phosphorylation of NS5A is believed to affect its biological
properties. However, NS5A lacks intrinsic protein kinase ac-
tivity, suggesting that its phosphorylation results from interac-
tion with one or more cellular kinases (10, 12–15, 25). Previous
in vitro studies demonstrated that CK2 and cAMP-dependent
protein kinase (PKA) are able to phosphorylate NS5A (12, 14).
Since NS5A performs a host of diverse functions, including the
modulation of several signal transduction pathways (6, 8, 28),
we expect that NS5A may be phosphorylated by additional
cellular kinases. To further investigate the phosphorylation of
NS5A, we have taken advantage of GST-yeast kinase fusion
proteins to screen the S. cerevisiae kinome for kinases that
phosphorylate NS5A in vitro.
The yeast genome is predicted to contain 124 protein ki-
nases, and 119 of these have been successfully expressed as
GST-kinase fusion proteins (36). Eight of these constructs
were capable of phosphorylating NS5A under the conditions
described here. Table 1 provides a summary of the distribution
by major groups of the GST-yeast kinases that phosphorylated
NS5A in vitro, and autoradiographs from representative sam-
ples are presented in Fig. 1. In order to eliminate the possibility
that phosphorylation would be influenced by the presence of a
tag used to facilitate protein purification, we used native NS5A
that was purified to homogeneity by sequential DEAE-Sepha-
rose and Mono Q anion-exchange chromatography (13).
hCK2, which phosphorylates NS5A in vitro (14), was used as a
positive control to demonstrate NS5A phosphorylation (Fig.
1B, lanes 14 and 28).
The yeast kinases that phosphorylated NS5A did so with
variable efficiencies. For example, comparison of lanes 2 and 3
demonstrates that the yeast kinase YPL204W (Fig. 1B, lane 3)
generated a stronger32P signal associated with NS5A phos-
phorylation than did construct YNL154C (Fig. 1B, lane 2).
This is not surprising, since these kinases are likely to have
different affinities for NS5A and may also have different re-
quirements for optimal activity. Because of the vast number of
kinases screened in this study, the phosphorylation reactions
were performed under identical conditions.
Several of the reactions revealed additional bands of phos-
phorylation that were presumably associated either with auto-
phosphorylation of the kinase or with contaminants copurify-
ing with the respective kinase. For example, although the
YOL128C construct was unable to phosphorylate NS5A, a
higher-molecular-weight band, presumably corresponding to
the autophosphorylated kinase, was detected (Fig. 1, lane 16).
TABLE 1. Distribution by major groups of the GST-yeast kinases
phosphorylating the HCV nonstructural protein NS5Aa
AGC .....................................................................YMR104C, YDR466W
aGST-protein kinase constructs representing 119 S. cerevisiae protein kinases
were purified by glutathione-agarose chromatography and subjected to an in
vitro kinase assay as described in Materials and Methods. Eight kinases were
shown to phosphorylate NS5A and are listed in their respective group in this
table. These kinases represent five major groups of yeast kinases: AGC (so
named because this group includes protein kinases A, G and C), CMGC (so
named because this group includes cyclin-dependent kinases, mitogen-activated
protein kinase, glycogen synthase kinase 3, and casein kinase 2), CK1 (so named
because this group includes casein kinase 1), STE (so named because this group
includes STE7, a protein kinase of the MAPK kinase family), CaMK (so named
because this group includes Ca2?/calmodulin-dependent kinases). The groups
are based in amino acid sequence homology of the known protein kinases. A
regular yeast kinase assay is described in Fig. 1.
3504 COITO ET AL.J. VIROL.
FIG. 1. Purity of the NS5A protein and in vitro phosphorylation of NS5A by yeast kinases. (A) The NS5A substrate used during this study was
tested for its purity. Native recombinant NS5A (1 ?g), purified from Sf9 cells, was resolved by SDS-PAGE and visualized by silver staining. The
arrow indicates the NS5A protein. (This panel is reprinted with the permission of the publisher of reference 13 and is shown here to demonstrate
the purity of the substrate, which was an essential prerequisite for our analyses.) (B) In a regular yeast kinase assay, baculovirus recombinant NS5A
purified from Sf9 cells (100 ng) was phosphorylated in kinase buffer containing GST-yeast kinase bound to glutathione beads as described in
Materials and Methods. The reaction was monitored by autoradiography of32P-labeled NS5A resolved by SDS-PAGE. Two autoradiography films
from two regular yeast kinase assays are shown. The arrows show the NS5A protein. Human recombinant casein kinase 2 was used as a positive
control to demonstrate phosphorylation of NS5A under the conditions described here. Yeast extracts from cells transfected with an empty
glutathione vector were used as a negative control to demonstrate that phosphorylation of NS5A was not associated with proteins nonspecifically
bound to either GST or the glutathione-agarose beads. The kinases present in the phosphorylation reaction were as follows: YDR283C (lane 1),
YNL154C (lane 2), YPL204W (lane 3), YDL101C (lane 4), YOL016C (lane 5), YOR061W (lane 6), YHR135C (lane 7), YMR104C (lane 8),
YGL059W (lane 9), YIL035C (lane 10), YBR028C (lane 11), YHR030C (lane 12), negative control (lane 13), positive control (lane 14), negative
control (lane 15), YOL128C (lane 16), YPL203W (lane 17), YPL236C (lane 18), YOR061W (lane 19), YDL108W (lane 20), YPL209C (lane 21),
YPL140C (lane 22), YPL026C (lane 23), YNL307C (lane 24), YBR059C (lane 25), YPL031C (lane 26), YNL154C (lane 27), and positive control
VOL. 78, 2004 SCREEN FOR KINASES THAT PHOSPHORYLATE NS5A3505
When the reaction was carried out in the presence of yeast
extracts from cells transfected with an empty glutathione vec-
tor, no phosphorylation of NS5A was detected (Fig. 1, lanes 13
and 15). These results demonstrate that phosphorylation of
NS5A is not associated with proteins nonspecifically bound to
either GST or the glutathione-agarose beads. In addition, the
yeast kinase GCN2, which has sequence homology with the
interferon-inducible protein kinase PKR, did not phosphory-
late NS5A (data not shown). This is consistent with our pre-
vious demonstrations that although NS5A can interact with
PKR, it is not phosphorylated by this kinase (6).
Identification of human protein kinases related to yeast
NS5A protein kinases. We used the sequences of the eight
yeast kinases (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi)
that phosphorylated NS5A to perform a National Center for
Biotechnology Information BLAST search (http://www.ncbi.n-
lm.nih.gov/BLAST) in order to identify human kinases with
sequence homology. The human kinases identified by this ap-
proach included candidates from the CK1, CMGC, STE,
CaMK, and AGC groups (Table 2). Consistent with previous
studies demonstrating that CK2 (14) and PKA (12) phosphor-
ylate NS5A, CK2 was identified as a top candidate for the
CMGC group, represented by the yeast constructs YIL035C
and YOR061W, and PKA was identified as a top candidate for
theAGC group, represented
YDR466W. These observations support the validity of this
approach and provided additional confidence that analysis of
the human protein kinases present on this list would serve as a
good starting point for subsequent in vitro NS5A phosphory-
lation studies. We therefore screened all the commercially
available kinases corresponding to the top five nonredundant
hits for each kinase group reported in Table 2, including CK2,
MEK1, MKK7?1, CHK2, AKT1, p70S6K, SGK1, and PDK1.
Because human kinase homologues were not available for CK1
delta and p90S6K, we screened the respective rat kinases.
In vitro phosphorylation of NS5A by human protein ki-
nases. Consistent with previous studies (14), we found that
hCK2 phosphorylated NS5A in vitro under the conditions de-
scribed here (Fig. 2A, lane 3). Similarly, rCK1 delta also phos-
phorylated NS5A (Fig. 2A, lane 5). It should be noted that a
faint lower-molecular-weight radiolabeled band was also de-
tected in the phosphorylation reaction with hCK2. Since this
band was also detected with hCK2 in the absence of NS5A in
the kinase assay (Fig. 2A, lane 2), we suspect that this corre-
sponds to phosphorylation of a contaminant present in the
We also found that NS5A was phosphorylated by members
of the STE group, including the mitogen-activated protein
kinase kinase (MAPK kinase) family members hMEK1 (Fig.
3A, lane 4), hMKK6 (Fig. 3B, lane 4), and hMKK7?1 (Fig. 3C,
lane 4). Although hMKK6 was not among the top five nonre-
dundant hits, we chose to screen this kinase because it is
nevertheless a closely related member of the MAPK family and
was readily available. For each kinase tested, hCK2 was used as
a positive control (Fig. 3A, B, and C, lanes 1). In each case, a
second phosphorylated band was detected. Analysis of the
kinases alone (Fig. 3A, B, and C, lanes 5) revealed that these
bands were detected in the absence of NS5A, demonstrating
that they are associated with the kinases. The observed molec-
ular weights are consistent with those expected for each kinase,
suggesting that these bands correlate to autophosphorylated
kinases. This idea is supported by previous reports describing
TABLE 2. Summary of the yeast kinases shown to phosphorylate NS5A and the related human kinases identified by sequence homologya
Yeast NS5A kinases
Related human kinases
YPL204WHRR25 CK1 CK1 delta isoform 1 (10?157), CK1 delta isoform 2 (10?157), CK1 epsilon
(10?156), CK1 alpha (10?149), CK1 gamma1 (10?95)
CK1 gamma 2 (7 ? 10?95), CK1 gamma 3 (5 ? 10?93), CK1 gamma 1 (5
? 10?92), CK1 alpha (5 ? 10?87), CK1 delta (10?83)
CK2 (10?163), GSK3 alpha (6 ? 10?17), GSK3 beta (6 ? 10?16), CDK-
like 2 (9 ? 10?14), CDK-like 5 (10?13)
CK2 (10?138), GSK3 beta (5 ? 10?17), GSK3 alpha (5 ? 10?17), CDK6
(4 ? 10?12), MAPK p38 beta (4 ? 10?11)
MEK1 (4 ? 10?50), MEK2 (8 ? 10?48), MEK5 (8 ? 10?42), MKK7? (4
? 10?39), MKK4 (4 ? 10?39)
CHK2 (6 ? 10?47), Ca2?/calmodulin-dependent kinase 1 (9 ? 10?46),
double cortin-like and Ca2?/calmodulin-dependent kinase-like 1 (5 ?
10?45), PKC mu (8 ? 10?36), PKD2 (8 ? 10?35)
SGK2 (3 ? 10?96), AKT3 (5 ? 10?82), p70S6K (4 ? 10?79), PKC theta
(2 ? 10?72), p90S6K (10?71)
AKT1 (10?65), PDK1 (10?65), p70S6K (3 ? 10?22), SGK1 (10?18), PKA
catalytic subunit beta (10?18)
YDR466W PKH1 AGC
aThe yeast kinases shown to phosphorylate NS5A are summarized here, along with the major groups to which the kinases belong. Also included are the top five
nonredundant hits resulting from database BLAST searches to identify related human protein kinases. These hits are sorted by the quality of the amino acid homology
between the yeast kinases and the human kinases from best to worst, with the expected value (e-value) indicated in parentheses. The expected value is an estimate of
the probability of a sequence occurring by random chance, given the BLAST database used for the search. A very low e-value indicates a good homology; a higher one
indicates a poor homology. Some of the related human kinases shown here are highly conserved with the respective yeast kinase, while others are not. For example,
the top five nonredundant hits to the yeast kinase YPL204C correspond to various CK1 species, all of which have high sequence homology (10?157to 10?95) with
YPL204W. A similar situation is observed for YNL154C. By contrast, the top five nonredundant hits to the yeast kinases YIL035C and YOR061W represent candidates
with a broader range of sequence homologies (10?163to 4 ? 10?11). MAPK p38, PKC, PKA, PKD, and SGK stand, respectively, for mitogen-activated protein kinase
p38, protein kinase C, cAMP-dependent protein kinase, protein kinase D, and serum/glucocorticoid-regulated kinase. The CK1, CMGC, STE, CaMK, and AGC
abbreviations are explained in Table 1.
bORF, open reading frame.
3506COITO ET AL. J. VIROL.
the autophosphorylation of these kinases (2, 3). However, we
cannot rule out the possibility that these bands represent con-
taminants in the kinase preparations.
Two of the top five candidates from the AGC kinase group,
hAKT1 (Fig. 3D, lane 2) and hp70S6K (Fig. 3E, lane 2),
phosphorylated NS5A in vitro. As was the case with kinases
from the STE group, a second band of32P-labeled material
that is likely to be an autophosphorylated form of the kinase
was also detected (Fig. 3D and 3E, lanes 1) (26, 32). The
remaining kinases from the AGC group (hPDK1, hSGK1, and
rp90S6K) did not phosphorylate NS5A (data not shown), nor
did hCHK2, the only commercially available kinase among the
top five nonredundant hits of the CaMK group (Fig. 3F, lane
4). However, a member of the CaMK group not among the top
five hits, Ca2?calmodulin-dependent kinase II, was able to
phosphorylate NS5A in vitro (data not shown). Although we
did not independently verify whether the kinases that failed to
phosphorylate NS5A were able to phosphorylate a known sub-
strate, the presence of a signal associated with autophosphor-
ylation suggests that these kinases were active.
In vitro phosphopeptide mapping of NS5A. The results de-
scribed above demonstrated the utility of a high-throughput
screen of the yeast kinome and revealed that human kinases
from several major kinase groups were able to phosphorylate
NS5A. In order to determine whether the sites of phosphory-
lation were unique for the different groups of kinases, we
compared the phosphopeptide maps of in vitro-phosphory-
lated NS5A. Figure 2B shows the tryptic/chymotryptic phos-
phopeptide maps of NS5A phosphorylated with either rCK1
delta or hCK2. One labeled phosphopeptide detected in the
presence of rCK1 delta, denoted by the arrow, was not de-
tected with hCK2, suggesting that the phosphorylation sites
may differ. Phosphopeptide maps generated with kinases from
the STE kinase group (i.e., MAPK family member MEK1 or
FIG. 2. In vitro phosphorylation of NS5A with CK1 delta and CK2. (A) Autoradiography of32P-labeled NS5A resolved by SDS-PAGE. The
CK2 used in this experiment is a human kinase, and the CK1 delta is a rat kinase. The phosphorylation reactions carried out were as follows: NS5A
minus kinase (lane 1), hCK2 minus NS5A (lane 2), hCK2 plus NS5A (lane 3), rCK1 minus NS5A (lane 4), and rCK1 plus NS5A (lane 5).
(B) Phosphopeptide mapping of NS5A phosphorylated with either rCK1 or hCK2. In vitro-phosphorylated NS5A was resolved by SDS-PAGE, and
the proteins were electroblotted onto nitrocellulose membranes. The radiolabeled NS5A bands were detected by autoradiography and subse-
quently sliced out and subjected to enzymatic digestion with trypsin and chymotrypsin. The peptides were applied on cellulose TLC plates at an
origin shown by the black circle. The peptides were then separated in the first dimension by one-dimensional electrophoresis (1st). The positively
charged peptides migrate toward the anode (negative plug [-]), and the negatively charged peptides migrate toward the cathode (the positive plug
[?]). Using the same TLC plate, the peptides were resolved in the second dimension (shown by the arrow labeled 2nd) by ascending chroma-
tography. The TLC plate was then exposed to film to generate the phosphopeptide map. The black dots show where the phosphopeptides are in
the map. Phosphorylation reactions carried out in the absence of NS5A (shown in this panel) or in the presence of NS5A but not the kinase (not
shown) were used as negative controls to test for peptides coming from potential contaminating proteins of molecular weight similar to that of
NS5A or poorly resolved kinases. The NS5A map of the phosphorylation reaction carried out in the presence of NS5A but not the kinase gives
an autoradiography without any signal (result not shown). The black arrow inside the CK1 panel shows the major NS5A peptide phosphorylated
by CK1 delta.
VOL. 78, 2004SCREEN FOR KINASES THAT PHOSPHORYLATE NS5A3507
MKK6) and the AGC kinase group (AKT1 and p70S6K) are
shown in Fig. 4 and 5, respectively. The phosphopeptide pat-
terns are quite distinct depending on the kinase used, demon-
strating that NS5A can serve as a substrate for a variety of
kinases, resulting in differential phosphorylation at multiple
serine and threonine residues in vitro. Phosphopeptide maps
were also generated from phosphorylation reactions carried
out in the absence of NS5A in order to monitor for the pres-
ence of contaminating phosphopeptides (negative control).
This was accomplished by cutting out and processing the same-
molecular-weight region of the nitrocellulose membrane from
lanes containing kinase only (see Materials and Methods). In
some instances, we detected a significant amount of radiola-
beled material in the absence of NS5A where no phosphopep-
tides were expected (i.e., negative control phosphopeptide
maps with hAKT1 or hp70S6K) (Fig. 5). We suspect that this
resulted from removal of some of the closely migrating kinase
when the region of the filter corresponding to the molecular
weight of NS5A was cut out for processing (see Fig. 3D and E,
lanes 2). NS5A phosphopeptides detected in vitro that match
with NS5A phosphopeptides detected in vivo are shown in Fig.
6 and discussed in more detail below.
In vivo phosphopeptide mapping of NS5A. In order to in-
vestigate the biological relevance of in vitro phosphorylation of
NS5A by the kinases identified above, we next examined the
phosphorylation of NS5A in cell culture. Briefly, COS-1 cells
were transfected with a plasmid encoding the isogenic NS5A
used for our in vitro studies, and in vivo-labeled NS5A was
generated using [32P]orthophosphate. Figure 6A shows an au-
toradiograph of NS5A immunoprecipitated with antibody spe-
cific to NS5A, resolved by SDS–10% PAGE, and transferred to
a nitrocellulose membrane. Consistent with previous findings
FIG. 3. In vitro phosphorylation of NS5A with members of the STE and AGC groups of human kinases. This figure shows the region of the
autoradiography film around the molecular weight of NS5A incubated with or without different kinases. The kinase reactions were carried out with
the related human kinases for the yeast kinases found to phosphorylate NS5A in this study. MEK1, MKK6, MKK7?1, AKT1, and p70S6K used
in this study are human recombinant proteins. Baculovirus recombinant NS5A purified from Sf9 cells (100 ng) was phosphorylated in kinase buffer
containing the appropriate human kinases as described under Materials and Methods. The reaction was monitored by autoradiography of
32P-labeled NS5A resolved by SDS-PAGE. In each experiment, hCK2 was used as a positive control, demonstrating that NS5A was phosphorylated
under the conditions used here. Arrows show NS5A or the kinase autophosphorylation. (A) Phosphorylation with hMEK1. Phosphorylation
reactions carried out with NS5A in the presence of either hCK2 (lane 1) or hMEK1 (lane 4) are shown. Negative controls including incubation
of NS5A (lane 2) or kinase alone (lanes 3 and 5) are shown for comparison. The arrows in this panel show two phosphoproteins, NS5A
phosphorylated by hCK2 or hMEK1 and the autophosphorylation band of MEK1. (B) Phosphorylation with hMKK6. Phosphorylation reactions
carried out with NS5A in the presence of either hCK2 (lane 1) or hMKK6 (lane 4) are shown. Negative controls including incubation of NS5A
(lane 3) or kinase alone (lanes 2 and 5) are shown for comparison. The arrows in this panel show two phosphoproteins, NS5A phosphorylated by
hCK2 or hMKK6 and the autophosphorylation band of MKK6. (C) Phosphorylation with hMKK7?1. Phosphorylation reactions carried out with
NS5A in the presence of either hCK2 (lane 1) or hMKK7?1 (lane 4) are shown. Negative controls including incubation of NS5A (lane 2) or kinase
alone (lanes 3 and 5) are shown for comparison. The arrows in this panel show two phosphoproteins, the NS5A phosphorylated by hCK2 or
hMKK7?1 and the autophosphorylation band of hMKK7?1. (D) Phosphorylation with hAKT1. Phosphorylation reactions carried out with NS5A
in the presence of either hCK2 (lane 5) or hAKT1 (lane 2) are shown. Negative controls including incubation of NS5A (lane 4) or kinase alone
(lanes 1 and 3) are shown for comparison. The arrows in this panel show two phosphoproteins, NS5A phosphorylated by hCK2 or hAKT1 and the
autophosphorylation band of hAKT1. (E) Phosphorylation with hp70S6K. Phosphorylation reactions carried out with NS5A in the presence of
either hCK2 (lane 5) or hp70S6K (lane 2) are shown. Negative controls including incubation of NS5A (lane 3) or kinase alone (lanes 1 and 4) are
shown for comparison. The arrows in this panel show three phosphoproteins, two bands for NS5A and one autophosphorylation band for hp70S6K.
The phosphorylation of NS5A by hp70S6K produced two phosphopeptides. (F) Phosphorylation with hCHK2. Phosphorylation reactions carried
out with NS5A in the presence of either hCK2 (lane 1) or hCHK2 (lane 4) are shown. Negative controls including incubation of NS5A (lane 5)
or kinase alone (lanes 2 and 3) are shown for comparison.
3508 COITO ET AL.J. VIROL.
(22, 30), two phosphorylated species of NS5A were detected.
The identity of these bands was confirmed by immunoblot
analysis with NS5A-specific monoclonal antibody (Fig. 6B). In
addition, a third higher-molecular-weight species (150 kDa)
was also detected during immunoprecipitation with cell lysates
expressing NS5A (Fig. 6A, lane 1). We suspect that this may
represent a binding partner of NS5A, since it was not detected
in cell lysates transfected with a control vector lacking NS5A
(Fig. 6A, lane 2). Each of the phosphorylated species of NS5A
was subsequently processed for two-dimensional phosphopep-
tide mapping. Since the phosphopeptide maps generated from
the two species of phosphorylated NS5A were nearly identical,
Fig. 6C shows the tryptic/chymotryptic phosphopeptide map
resulting from the lower-molecular-weight species. Immuno-
FIG. 4. Phosphopeptide mapping of NS5A phosphorylated in vitro with either hMEK1 or hMKK6. NS5A was phosphorylated with either the
hMEK1 or hMKK6 kinase from the MAPK kinase family and subjected to phosphopeptide mapping as described in the legend to Fig. 3. The
negative control panel is the analysis of the phosphopeptide signal from the phosphorylation reactions using hMEK1 or hMKK6 carried out in the
absence of NS5A. The dotted circles labeled with letters are in vitro-labeled NS5A phosphopeptides that match with the NS5A phosphopeptides
generated in vivo (see Fig. 6B).
FIG. 5. Phosphopeptide mapping of NS5A phosphorylated in vitro with either hAKT1 or hp70S6K. NS5A was phosphorylated with either the
hAKT1 or hp70S6K kinase and subjected to phosphopeptide mapping as described in the legend to Fig. 3. The negative control panel is the analysis
of the phosphopeptide signal from the phosphorylation reactions using hAKT1 or hp70S6K carried out in the absence of NS5A. The three black
arrows inside the phosphopeptide map of NS5A phosphorylated by hp70S6K show negatively charged NS5A phosphopeptides, which run toward
the positive plug during the first dimension of the phosphopeptide map. The dotted circles labeled with letters are in vitro-labeled NS5A
phosphopeptides that match the NS5A phosphopeptides generated in vivo (see Fig. 6B).
VOL. 78, 2004 SCREEN FOR KINASES THAT PHOSPHORYLATE NS5A3509
FIG. 6. Phosphopeptide mapping of NS5A phosphorylated in vivo. COS-1 cells were transiently transfected with 2 or 10 ?g of plasmid encoding
isogenic NS5A-1b used in this study for the in vitro NS5A maps. As a negative control, COS-1 cells were transfected with the empty plasmid vector.
At 15 h posttransfection, cells were labeled with32P for 4 h at 37°C and harvested, and an antibody against NS5A was added for 3 h. The proteins
associated with the NS5A antibody were isolated on protein G-agarose beads for 2 h at 4°C. The samples were immediately eluted with protein
sample buffer, analyzed by SDS-PAGE (10% gel), and transferred to a nitrocellulose membrane. (A) An autoradiograph of the nitrocellulose
membrane that contains the proteins isolated with the NS5A antibody and the protein G beads. (B) Immunoblot analysis of NS5A expressed in
COS-1 cells. Nonradioactive cell lysates (200 ng) from cells transfected with 10 ?g (lane 1), 2 ?g (lane 2), or 0 ?g (lane 3) of pcDNA3 NS5A-1b
were resolved by SDS-PAGE (10% gel) and transferred to a nitrocellulose membrane prior to blotting with an NS5A-specific monoclonal antibody.
3510 COITO ET AL.J. VIROL.
precipitates from protein lysates of COS-1 cells transfected
with a control vector lacking NS5A were run in parallel, and
the same-molecular-weight region of the nitrocellulose mem-
brane was processed to monitor for the presence of contami-
nating phosphopeptides (Fig. 6C, negative control). Phos-
phopeptides unique to NS5A, as determined by comparison
with the negative control, are labeled a to g.
The autoradiography films from the in vivo and in vitro
phosphopeptide maps were then overlaid for comparison. The
comigrating spots observed in the in vitro mapping experi-
ments are given the same letter as for the in vivo map (com-
pare Fig. 4 and 5 with Fig. 6). Interestingly, six of the seven
phosphopeptides (labeled a to f) detected in vivo were also
detected during in vitro phosphorylation of NS5A with
hp70S6K (Fig. 5). Four of these phosphopeptides (labeled b, d,
e, and f) were also detected when NS5A was phosphorylated
with hAKT1 (Fig. 5). By contrast, only a small number of the
peptides detected in vivo could also be seen during in vitro
phosphorylation with either MEK1 or MKK6 (Fig. 4, phos-
phopeptide c for MEK1; phosphopeptides b, c, and d for
One in vivo phosphopeptide (labeled g) was not detected in
any of the in vitro phosphopeptide maps presented here. It is
possible that this phosphopeptide resulted from the phosphor-
ylation of NS5A by another kinase not characterized by two-
dimensional phosphopeptide mapping in this study. Last, it is
important to note that the in vivo phosphopeptide map re-
ported here is more complex than what we previously observed
(13). The conditions used here for enzymatic digestion of
NS5A were more aggressive than those used in our previous
study and more closely reflect those used to generate the dis-
tinct and complex phosphopeptide patterns reported for
NS5A-1a (25). Therefore, we suspect that the increased com-
plexity of the phosphopeptide maps observed for NS5A-1b is
attributable to more complete enzymatic digestion.
Inhibition of the mTOR/p70S6K pathway using rapamycin.
One of the most interesting and important kinases identified by
our approach was p70S6K. It is well described in the literature
that the mammalian target of rapamycin (mTOR) pathway is a
major pathway activating p70S6K (21, 29, 33) and that rapa-
mycin (21, 29, 33) is commonly used to study p70S6K activity.
We therefore used rapamycin to investigate the in vivo contri-
bution of p70S6K to the phosphorylation of NS5A. We already
observed that on a one-dimensional SDS-PAGE gel, the phos-
phorylation of NS5A was significantly reduced by addition of
rapamycin (data not shown), suggesting that the p70S6K acti-
vated by mTOR could play a role in the phosphorylation of
NS5A. We then analyzed the phospho-NS5A-peptide map ob-
tained from cells treated with or without rapamycin, and as
shown in Fig. 6D, the phosphorylation of three of the major in
vivo NS5A peptides phosphorylated by p70S6K (spots b, d, and
f) was significantly reduced. However, as shown in Fig. 6D,
rapamycin does not eliminate all the NS5A phosphopeptides
(for example, spots a, c, and g), which suggests that this inhib-
itor is acting with specificity. This provides additional critical
evidence that p70S6K and potentially closely related members
phosphorylate NS5A in vivo.
Based on our findings, we suggest that hp70S6K is a human
kinase responsible for phosphorylating NS5A in vivo. An ex-
amination of the sequence of the NS5A-1b species used here
revealed sequences closely related to the consensus phosphor-
ylation motifs of p70S6K (K/R-X-R-X-X-S/T-X). In addition, a
slight variation in this motif is present around serine 2194, a
highly conserved major phosphorylation site in NS5A-1b and
in NS5A proteins from other HCV genotypes (13). In contrast,
we were unable to detect this motif near serine 2321, a major
phosphorylation site in the HCV 1a genotype (24). Further
experiments using specific inhibitors or RNA interference
(RNAi) strategies aimed at inhibiting or down-regulating the
expression of the various kinases identified may provide fur-
ther insight into the relative contributions of these kinases to
the in vivo phosphorylation of NS5A.
Conclusions. It is likely that phosphorylation of NS5A plays
a role in regulating the diverse functions of this protein. In this
regard, we have been particularly interested in understanding
the significance of NS5A phosphorylation in the ability of HCV
to interfere with cellular signaling. For example, the interac-
tion between NS5A and Grb2 may inhibit the MAPK pathway,
resulting in down-regulation of cellular mRNA translation
(22). In addition, NS5A plays a role in regulating viral mRNA
translation initiation from the HCV internal ribosome entry
site (9). Given the ability of NS5A to regulate mRNA trans-
lation, it is intriguing that NS5A was phosphorylated by both
hAKT1 and hp70S6K, cellular kinases that themselves play
important roles in translational control (1, 31, 34). Phosphor-
ylation of NS5A by AKT and p70S6K could also induce HCV
internal ribosome entry site-mediated translation, thereby fa-
cilitating HCV replication and persistence.
The two differentially phosphorylated species of NS5A are indicated by arrows. In addition, several nonspecifically reacting bands were detected,
as evidenced by their presence in cell lysates in the presence or absence of transfection with pcDNA3 NS5A-1b. (C) In vivo phosphopeptide map
of NS5A. Due to the clear resolution of the two NS5A bands, each band was analyzed individually. The autoradiograph of the map of the bottom
NS5A band is shown here. As a negative control, the same-molecular-weight region of the immunoprecipitation from cells transfected with an
empty plasmid was subjected to phosphopeptide mapping. Regular phosphopeptide mapping was done as described in Materials and Methods and
in the legend to Fig. 3. The dotted circles show the specific in vivo NS5A phosphopeptides (labeled a to g). Each letter corresponds to a different
phosphopeptide. This in vivo NS5A phosphopeptide autoradiograph was superimposed on each in vitro NS5A phosphopeptide map obtained in
this study. The spots matching between the in vivo and in vitro maps have the same letters. (D) In vivo phosphopeptide map of NS5A expressed
in COS-1 cells inhibited or not inhibited by rapamycin. This panel shows the phospho-NS5A-peptide map from cells inhibited or not inhibited with
rapamycin. The autoradiograph of the map of the bottom NS5A band is shown here. The band from the same region of the immunoprecipitation
from cells transfected with an empty plasmid and inhibited or not inhibited with rapamycin was subjected to phosphopeptide mapping and showed
the same phosphopeptide pattern as the one seen in the negative control of panel C. The dotted circles show the localization of the phosphopep-
tides (labeled a to g) observed in the NS5A map in panel C. The ratios of the radioactivity counts between NS5A minus rapamycin and NS5A plus
rapamycin for each phosphopeptide in the NS5A map in panel C have been calculated. These ratios are the following: a, 0.9; b, 5.0; c, 0.9; d, 11.5;
e, 0.6; f, 4.4; g, 1.1. These ratios represent all experiments performed.
VOL. 78, 2004 SCREEN FOR KINASES THAT PHOSPHORYLATE NS5A3511
Clearly, the complexity of the in vivo phosphorylation pat-
terns observed here warrants further experimentation to de-
termine the significance of these findings. It is likely that mul-
tiple kinases, in addition to p70S6K, contribute to NS5A
phosphorylation, including AKT, MEK1, and MKK6. In addi-
tion, although it is possible to positively identify kinases that
phosphorylate NS5A in vivo, it is more difficult to rule out the
possibility that other kinases may also contribute to NS5A
phosphorylation. In this regard, it is also important that some
of the kinases that phosphorylated NS5A in vitro may have no
relevance in vivo and that in vitro assays may not detect bio-
logically relevant NS5A kinases that require additional regu-
latory factors normally present in vivo. Nevertheless, our ap-
proach identified several kinases capable of phosphorylating
NS5A. This information should facilitate the identification of
the protein kinases that phosphorylate NS5A in liver cells, the
natural site of HCV replication (7). Further studies aimed at
evaluating the role of the identified NS5A kinases using in vivo
model systems, such as the HCV replicon (18), may provide a
better understanding of the role of NS5A phosphorylation
during the HCV life cycle.
In summary, this study illustrates the use of a kinome-scale
high-throughput screening approach for identifying protein ki-
nases of interest. We propose that this approach represents an
ideal model for other studies aimed at identifying protein ki-
nases for further investigation of functionally relevant phos-
phorylation events. The development of similar approaches for
characterizing gene function directly at the protein level should
contribute significantly to the success of efforts in functional
genomics, and such approaches may be generally applicable to
the high-throughput purification and biochemical analysis of
other proteins by mechanistic class.
This work was supported by Public Health Service grant R01-
AI47304 from the National Institute of Allergy and Infectious Diseases
and grant P30DA015625 to the Center for Functional Genomics &
HCV-Associated Liver Disease from the National Institute on Drug
We thank Eric M. Phizicky at the University of Rochester, Roches-
ter, N.Y., for providing us with the S. cerevisiae protein kinase con-
structs cloned in the plasmid pYEX4T-1 and expressed in the EJ758
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