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J Biol Chem, Vol. 273, Issue 45, 29879-29887, November 6, 1998
From the The human immunodeficiency virus type 1 (HIV-1)
Vif protein plays a critical role in virus replication and infectivity.
Here we show that Vif is phosphorylated and regulated by p44/42
mitogen-activated protein kinase (MAPK). Vif phosphorylation by MAPK
was demonstrated in vitro as well as in vivo
and was shown to occur on serine and threonine residues.
Two-dimensional tryptic phosphopeptide mapping indicated that Vif is
phosphorylated by MAPK on the same sites in vitro and
in vivo. Radioactive peptide sequencing identified two
phosphorylation sites, Thr96 and Ser165. These
phosphorylation sites do not correspond to the known optimum consensus
sequences for phosphorylation by MAPK (PX(S/T)P) nor to the
minimum consensus sequence ((S/T)P), indicating that MAPK can
phosphorylate proteins at sites other than those containing the
PX(S/T)P or (S/T)P motifs. Synthetic Vif peptides
corresponding to the local sequences of the phosphorylation sites were
not phosphorylated by MAPK, suggesting that recognition of these sites
by MAPK is likely to require structural determinants outside the
phosphorylation site. Mutations of the Thr96 site, which is
conserved among Vif sequences from HIV-1, HIV-2, and SIV, resulted in
significant loss of Vif activity and inhibition of HIV-1 replication.
These results suggest that MAPK plays a direct role in regulating HIV-1
replication and infectivity by phosphorylating Vif and identify a novel
mechanism for activation of HIV-1 replication by mitogens and other
extracellular stimuli.
The Vif protein of human immunodeficiency virus type 1 (HIV-1)1 and other
lentiviruses is essential for virus replication in peripheral blood T
lymphocytes in vitro and in vivo (1-5). Vif enhances the infectivity of HIV-1 virions as much as 50-1000-fold (6-8). In the absence of Vif, virions can initiate reverse
transcription but cannot complete proviral DNA synthesis (2, 8-11).
This reflects an effect of Vif during virus production, since the
defect can be complemented by Vif expression in the producer cell but not in the target cell (1-3, 8). The biochemical mechanism and
regulation of Vif function are unknown. Several lines of evidence suggest that Vif plays a role in virus assembly (1, 2, 8, 11, 12). Vif
mutant virions exhibit a structural abnormality in the virion core (4,
13), raising the possibility that one of the Gag or Pol proteins may be
a target for the action of Vif. Some immortalized cell lines do not
require Vif to produce fully infectious virus (1-3, 8, 9), suggesting
that Vif may compensate for a cell-specific factor or neutralize an
inhibitory factor that interferes with correct virus assembly. Membrane
association of Vif is important for its biological activity and appears
to be mediated by an interaction between basic domains in the C
terminus and a membrane-associated protein (14).
We previously demonstrated that Vif is phosphorylated on serine and
threonine residues by a cellular kinase(s) (15). Vif phosphorylation
was shown to occur on Ser144, Thr155, and
Thr188. Mutation of Ser144, which is contained
within the most highly conserved motif in Vif proteins from all
lentiviruses, resulted in significant loss of Vif activity and
inhibited HIV-1 replication, suggesting that phosphorylation at this
site plays an important role in regulating virus replication and
infectivity. The kinase(s) that phosphorylates Vif was not identified.
The Vif kinase(s) was relatively insensitive to inhibitors of protein
kinase C, cAMP-dependent kinase, and cGMP-dependent kinase, suggesting that it is distinct from
these enzymes.
A critical aspect of HIV-1 replication in T lymphocytes is activation
by mitogens and other extracellular stimuli (16). However, little is
known about the signal transduction pathways that activate HIV-1
replication in response to extracellular stimulation. Previous studies
have shown that the HIV-1 p17 Gag, p24 Gag, Vif, Vpu, Rev, and Nef
proteins are phosphorylated by cellular kinases in vitro and
in vivo (15, 17-26). p17 Gag, Nef, and Rev are
phosphorylated on serine/threonine residues by protein kinase C (17,
19, 22), and Vpu is phosphorylated on serine by casein kinase II (23).
However, many kinases that phosphorylate and regulate the functions of
HIV-1 proteins have not been identified.
In this report, we show that HIV-1 Vif is phosphorylated on
Thr96 and Ser165 by p44/42 mitogen-activated
protein kinase (also known as ERK1 and ERK2, hereafter referred to as
MAPK) both in vitro and in vivo. These sites do
not correspond to the known consensus sites for MAPK substrates,
indicating that MAPK can phosphorylate proteins at sites other than
those containing the PX(S/T)P or (S/T)P motifs. Mutations of
the Thr96 site, which is highly conserved among Vif
sequences from HIV-1, HIV-2, and SIV, result in loss of Vif activity
and inhibition of HIV-1 replication, suggesting that phosphorylation at
this site is likely to be important for Vif activity. These results provide a direct link between MAPK and the regulation of HIV-1 replication and infectivity. This mechanism may contribute to the
activation of HIV-1 replication by mitogens and other extracellular stimuli.
Materials--
Rabbit anti-ERK1 and anti-ERK2 were from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-phosphorylated
p44/42 MAPK was from New England Biolabs. Recombinant murine p42 MAPK was from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant Vif
protein derived from the HXB2 HIV-1 isolate and containing a histidine
tag at the N terminus was expressed in Escherichia coli
MC10611 containing the Vif expressor plasmid pD10Vif and purified using
a Ni2+-NTA-agarose column as described (15). The pMEK-R4F
plasmid (27, 28), a constitutively activated kinase expressor plasmid for mutant MAPK kinase (MEK) (also called Cell Culture--
The T cell lines CEM, SupT1, and H9 were
maintained in RPMI medium containing 10% fetal calf serum. COS-1,
HeLa, and 293T cells were maintained in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum. For cell stimulation with PMA,
EGF, bovine serum albumin, or serum, cells were serum-starved for
24 h and then stimulated for 15 min.
In Vitro Phosphorylation Assays--
Recombinant Vif protein was
used as a substrate for in vitro phosphorylation assays as
described (15). Phosphorylation by MAPK was performed using
immunoprecipitated MAPK or recombinant p42 MAPK. Cell lysates were
prepared in lysis buffer (10 mM Tris-HCl, pH 7.5, 1.0%
Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 50 mM NaF, 50 mM KF, 25 mM imidazole,
0.6 mM sodium orthovanadate, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml antipain, and 5 µg/ml pepstatin). Lysates were cleared by centrifugation at
12,000 × g for 10 min at 4 °C, and the supernatants
were immunoprecipitated with anti-ERK1 and anti-ERK2 (5 µl of each).
The MAPK immunocomplexes were isolated using protein A-Sepharose and
washed twice with lysis buffer, twice with lysis buffer containing 0.5 M NaCl, and once with kinase buffer (10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 0.5 mM dithiothreitol) prior to
resuspension in 50 µl of kinase buffer. An aliquot of
immunoprecipitated MAPK (12.5 µl), recombinant p42 MAPK (50 ng), or
CEM cell lysate (2 µg) was incubated for 30 min at room temperature
with kinase reaction mixture (2 µg of MBP or 2 µg of recombinant
Vif protein, 20 µM ATP, and 2.5-5.0 µCi of
[ In Vivo Phosphorylation of Vif in Intact Cells--
Vif was
expressed in 293T cells using a vaccinia virus expression system as
described (15). Vif containing a histidine tag at the N terminus was
expressed from pTM1/hVif under control of the T7 promoter. 293T cells
at 80% confluence in 60-mm plates were infected with vaccinia virus
VV-T7 containing the T7 polymerase gene in serum-free Dulbecco's
modified Eagle's medium for 1 h and then transfected by
lipofection with 3.75 µg of pTM1/hVif and 3.75 µg of pMEK-R4F (27,
28) or control vector pSG5 (Stratagene) using 22.5 µg of DOTAP
(Boehringer Mannheim) according to the manufacturer's instructions. At
10 h after transfection, the medium was removed and replaced with
the same medium containing 0.5% fetal calf serum after washing the
cells with phosphate-buffered saline. At 36 h after transfection,
cells were labeled with [32P]orthophosphate at 1 mCi/ml in phosphate-free medium containing 0.5% dialyzed fetal calf
serum for 2 h. Cells were washed with ice-cold phosphate-buffered
saline and lysed with 6 M guanidine HCl, 0.1 M
sodium phosphate, 20 mM imidazole, pH 8.0. Lysates were
centrifuged at 100,000 × g for 40 min, and the
supernatants were mixed with 30 µl of Ni2+-NTA-agarose.
Vif was then purified as described (15), resolved by SDS-PAGE,
transferred to PVDF membrane, and visualized by autoradiography or
immunoblotting with rabbit anti-Vif. MAPK activation was detected by
immunoblotting with anti-phosphorylated MAPK, which detects p44/42 MAPK
only when activated by phosphorylation.
In-gel Kinase Assays--
In-gel kinase assays were carried out
essentially as described (29, 30). Briefly, approximately 20 µg of
CEM cell lysate was subjected to electrophoresis on a 10%
SDS-polyacrylamide gel containing 500 µg of recombinant Vif/ml or 200 µg of MBP/ml co-polymerized in the separating gel. After
electrophoresis, the gel was washed twice with buffer A (50 mM Tris-HCl, pH 8.0, 20% isopropyl alcohol), once with
buffer B (50 mM Tris-HCl, pH 8.0, and 5 mM
Two-dimensional Tryptic Phosphopeptide
Mapping--
Two-dimensional tryptic phosphopeptide mapping was
performed as described (15). Recombinant Vif was phosphorylated by
recombinant p42 MAPK or by MAPK immunoprecipitated from PMA-stimulated
HeLa cells with anti-ERK1 and anti-ERK2 as described above in the
presence of [ Phosphoamino Acid Analysis and Identification of Phosphorylation
Sites--
Phosphoamino acid analysis was performed as described (15).
To identify phosphorylation sites, recombinant Vif was phosphorylated by recombinant p42 MAPK as described above. Proteolytic digestion with
sequencing grade trypsin (Boehringer Mannheim) and radioactive peptide
sequencing were then performed as described (15). Briefly, the
32P-labeled Vif was digested with trypsin in 50 mM NH4HCO3 for 15 h at
37 °C. The peptides were resolved by reverse phase HPLC using a
linear gradient from 5 to 100% acetonitrile in 0.1% trifluoacetic acid. Fractions (30 s) were collected and analyzed by Cerenkov counting. The molecular masses of the radioactive peptides were determined by laser-assisted desorption mass spectrometry using a
Lasermat mass spectrometer (Finnigan Mat Ltd., Hemel Hempsted, United Kingdom) and the sequences were confirmed by limited
N-terminal peptide sequencing (15).
Immunoblotting--
Cells were washed twice with
phosphate-buffered saline, lysed with ice-cold buffer (50 mM Tris-HCl, pH 7.5, 1.0% Triton X-100, 150 mM
NaCl, 25 mM NaF, 25 mM KF, 12.5 mM
imidazole, 0.3 mM sodium orthovanadate, 0.5 mM
EGTA, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 25 µg/ml
antipain, and 2.5 µg/ml pepstatin). The lysates were freeze-thawed
once and then centrifuged for 10 min at 4 °C at 13,000 × g. Samples with equivalent amounts of protein (5-10 µg)
were resolved by 15% SDS-PAGE and transferred to PVDF membrane for
immunoblotting with rabbit anti-ERK1 or anti-ERK1 and anti-ERK2 (1:1500
dilution of each), rabbit anti-phosphorylated MAPK (1:1000 dilution),
or rabbit anti-Vif (1:2000 dilution) (15) using the ECL system (Amersham Pharmacia Biotech) as described (15).
Analysis of Vif Mutants T96A and T96E--
Vif mutants were
generated in the SphI-SalI fragment of the pHXB2
plasmid, which contains full-length HIV-1 proviral DNA (1) subcloned
into pALTER-1 (Promega). The double-stranded plasmid was used as the
template for site-directed mutagenesis with primers containing the
desired mutations and a new HincII site (underlined sequences) (5'-GTT CAG GGT CAA CTT GTG CGC TAT ATC TC-3'
and 5'-GTT AAG GGT CAA CTT GTT CGC ATAT ATC TC-3') (Altered
Sites II In Vitro Mutagenesis Kit, Promega). The
SphI-SalI fragment containing the desired
mutations was subcloned into pHXB2. A similar method was used to
generate the S144A Vif mutant. The ability of the Vif mutant viruses to
replicate was determined by transfection of H9, CEM, or SupT1 cells
(107 cells) with 10 µg of the wild-type or mutant pHXB2
plasmids by the DEAE-dextran method followed by culturing the cells in
medium and monitoring reverse transcriptase (RT) activity in the
culture supernatants as described (1). Equal transfection efficiencies were confirmed by cotransfecting the pGL3 control vector plasmid (Promega) and normalizing for luciferase activity (Luciferase Assay
System, Promega). The infectivity of virions produced by H9 cells
transfected with the pHXB2 plasmids was determined by infection of
fresh H9 cells with equivalent amounts of virus (20,000 RT units) for
16 h at 37 °C. The cells were then washed twice and cultured in
fresh medium, and RT activity in the culture supernatants was
quantitated on day 11 after infection. Data were analyzed by analysis
of variance with post hoc Tukey-Kramer test and are expressed as means ± S.D.
Phosphorylation of Vif by MAPK--
To identify Vif kinase(s) in
cell lysates, we used Vif as a substrate for in-gel kinase assays. CEM
cell lysates were resolved by electrophoresis on an SDS-polyacrylamide
gel co-polymerized with recombinant Vif. After denaturation and
renaturation of the proteins in the gel, in-gel kinase activity was
determined by incubating the gel in kinase buffer containing
[
We then performed in vitro kinase assays to examine
phosphorylation of Vif by recombinant p42 MAPK or by MAPK
immunoprecipitated from stimulated cells. We found that the 24-kDa Vif
protein was highly phosphorylated on serine and threonine residues by
recombinant MAPK or by immunoprecipitated MAPK (Fig.
2). Vif phosphorylation by
immunoprecipitated MAPK was dramatically activated by stimulation of
serum-starved COS-1 cells with serum, PMA, EGF, or bovine serum albumin, which activate MAPK in many different types of cells (31, 32)
(Fig. 2A and data not shown). Similar results were obtained
using MBP as the MAPK substrate (Fig. 2B). Stimulation by
these MAPK activators did not significantly alter the expression of
MAPK (Fig. 2C). Kinetic studies showed that phosphorylation of Vif by MAPK was linear within the first 30 min and then reached a
maximum level at 45 min (Fig.
3A). The Km
for Vif phosphorylation by recombinant MAPK was approximately 1.6 µM (Fig. 3B), which is approximately 10-fold
lower than that obtained using MBP as the substrate (33). These results
demonstrate that HIV-1 Vif is directly phosphorylated by MAPK.
To determine whether Vif is phosphorylated by MAPK in vivo,
Vif was expressed in 293T cells using a recombinant vaccinia virus expression system. 293T cells were infected with recombinant vaccinia virus harboring a T7 RNA polymerase gene and then co-transfected with a
Vif expressor plasmid that contains a T7 promoter and pMEK-R4F, a
constitutively activated kinase expressor plasmid for mutant MAPK
kinase (MEK) (27, 28), the MAPK activating enzyme, or control vector
plasmid. MAPK kinase activates and phosphorylates p44/42 MAPK, but not
the c-Jun N-terminal kinase/stress-activated protein kinase or p38/HOG
MAPKs (34). The transfected cells were metabolically labeled with
[32P]orthophosphate, and the histidine-tagged Vif protein
was isolated by binding to Ni2+-NTA-agarose. The
radiolabeled proteins were separated by SDS-PAGE, transferred to PVDF
membrane, and detected by autoradiography or immunoblotting (Fig.
4). To confirm MAPK activation by
co-expression of constitutively activated MAPK kinase, MAPK activity
was determined by immunoblotting with an anti-phosphorylated MAPK
antibody that detects p44/42 MAPK only when activated by
phosphorylation. As expected, co-expression of constitutively activated
MAPK kinase induced MAPK activation (Fig. 4A), but did not
significantly affect the level of Vif expression (Fig. 4B).
[32P]orthophosphate labeling of Vif was markedly
stimulated by co-expression of constitutively activated MAPK kinase
(Fig. 4C). Phosphoamino acid analysis of Vif phosphorylated
in cells co-expressing constitutively activated MAPK kinase showed that
phosphorylation occurred on serine and threonine residues (Fig.
4D).
The preceding experiment suggests that Vif is phosphorylated by MAPK
in vivo. To confirm this possibility, we compared the two-dimensional tryptic phosphopeptide maps of Vif phosphorylated in vitro by recombinant p42 MAPK or by MAPK
immunoprecipitated from cells stimulated with PMA and Vif
phosphorylated in vivo in intact cells co-expressing
constitutively activated MAPK kinase. Three major tryptic
phosphopeptides generated from Vif phosphorylated in vitro
by recombinant MAPK or by immunoprecipitated MAPK comigrated with three
major phosphopeptides generated from Vif phosphorylated in
vivo in cells co-expressing constitutively activated MAPK kinase (Fig. 5, A, B, and
D, spots 1-3). Two minor
phosphopeptides were also shown to comigrate (spots
4 and 5). Comigration of the tryptic phosphopeptides was confirmed by mixing experiments (Fig. 5,
C and D). Three phosphopeptides generated from
Vif phosphorylated in vivo in cells co-expressing
constitutively activated MAPK kinase were not generated from Vif
phosphorylated in vitro by recombinant p42 MAPK
(spots 6 and 7 and data not shown).
These results demonstrate that Vif is phosphorylated by MAPK as well as
other kinases in vivo and indicate that Vif phosphorylation
by MAPK occurs on the same sites in vitro and in
vivo.
Identification of MAPK Phosphorylation Sites in Vif--
The
preceding experiments suggest that Vif is phosphorylated by MAPK on at
least three sites. We previously identified three major phosphorylation
sites in Vif (Ser144, Thr155, and
Thr188) (15). Two-dimensional tryptic phosphopeptide
mapping studies indicated that at least three additional sites are
phosphorylated in vivo. To identify the phosphorylation
sites in Vif used by MAPK, recombinant Vif was phosphorylated in
vitro by recombinant p42 MAPK and digested by trypsin. The tryptic
peptides were then separated by reverse-phase HPLC (Fig.
6A). Two major radioactive peaks were identified (Fig. 6B), in addition to several
minor radioactive peaks. The first major radioactive peak (peptide I) contained only phosphoserine (Fig. 6C). The second major
peak (peptide II) contained only phosphothreonine (Fig. 6C).
The two radioactive peptides were analyzed by laser-assisted desorption mass spectrometry to determine their molecular masses and by limited N-terminal sequencing. Identification of the peptide sequences (Table
I) showed that peptide I corresponds to
the amino acid sequence at positions 159-168 and peptide II
corresponds to the sequence at positions 94-122. Peptide I contains
only one serine residue, and peptide II contains only one threonine
residue (Table I). These results together with the phosphoamino acid
analysis of the two radioactive peptides (Fig. 6C)
demonstrate that Vif is phosphorylated by MAPK on Thr96 and
Ser165 (Table I). These phosphorylation sites do not
correspond to the known optimum consensus sequence for phosphorylation
by MAPK (PX(S/T)P), nor to the minimum consensus sequence
((S/T)P) identified in previous studies (32, 35, 36). Based on these
findings, we used synthetic peptides corresponding to the local Vif
sequences surrounding the Thr96 or
Ser165/Thr167 phosphorylation sites as
substrates for in vitro phosphorylation by recombinant p42
MAPK. Neither Vif peptide was phosphorylated by recombinant MAPK, while
the positive control MBP peptide was highly phosphorylated (Fig.
6D). These results suggest that the phosphorylation sites in
Vif can be used as MAPK substrates only within the context of the
full-length protein.
Replication of Thr96 Vif Mutants--
The
Thr96 phosphorylation site identified in the preceding
experiments is highly conserved among Vif sequences of HIV-1, HIV-2, and SIV. The Ser165 phosphorylation site is conserved in
HIV-1, but not in HIV-2 or SIV sequences. To determine whether MAPK
phosphorylation of Vif on Thr96 is important for its
biological activity, this residue was replaced with Ala or Glu. The
T96E mutation was analyzed to determine whether a negatively charged
residue could substitute for phosphothreonine. The T96A and T96E
mutations were introduced into plasmids containing HIV-1 proviral DNA,
and their effects on HIV-1 replication were examined in three different
cell lines. A mutation of Ser144 to alanine was introduced
into an HIV-1 proviral DNA plasmid to permit simultaneous comparison of
the Thr96 mutants with the S144A mutant, since
phosphorylation at this site is likely to be important for Vif function
(15). Vif is required for HIV-1 replication in primary lymphocytes and
some T cell lines (1-3, 8, 9). The wild-type and Vif mutant HIV-1
proviral DNAs were transfected into H9 cells, which require Vif
function for HIV-1 replication (1, 2, 4). Virus replication was
determined by measuring RT activity in the culture supernatants. Replication of the Thr96 and Ser144 Vif mutant
viruses was significantly impaired in H9 cells relative to the
wild-type (p < 0.001 at day 14 by analysis of variance with post hoc Tukey-Kramer test) (Fig.
7A). The T96E mutation resulted in more significant impairment of virus replication than the
T96A and S144A mutations, indicating that placing a negatively charged
residue at this position does not restore Vif function. The wild-type
and Vif mutant proviral DNAs were then transfected into CEM cells,
which can support low levels of HIV-1 replication in the absence of Vif
(1). The T96A, T96E, and S144A mutations significantly impaired HIV-1
replication in CEM cells (p < 0.01 at day 16) (Fig.
7A). In contrast, these mutations had no effect on virus
replication in SupT1 cells, which do not require Vif for HIV-1
replication (1, 2). Thus, the Thr96 and Ser144
mutations affected HIV-1 replication only in cell lines that require
Vif. Immunoblotting of cell lysates from HIV-1-infected CEM cultures
with rabbit anti-Vif serum (15) showed that expression of the T96A,
T96E, and S144A Vif mutant proteins was similar to that of the
wild-type protein (data not shown). The T96A, T96E, and S144A mutations
significantly reduced the infectivity of HIV-1 virions produced by
transfected H9 cells (p < 0.001) (Fig. 7B). These results suggest that MAPK phosphorylation of Vif on
Thr96 is important for Vif function and HIV-1 replication.
The additional finding that the Ser144 phosphorylation site
is important for HIV-1 replication and infectivity is consistent with
our previous studies of the S144A mutant (15).
ERK1 and ERK2 MAPK are serine/threonine kinases that are present
in all cell types and play a critical role in the regulation of cell
proliferation and differentiation in response to mitogens and a wide
variety of growth factors and cytokines (reviewed in Refs. 31, 32, 37,
and 38). Upon activation, these closely related MAPK isoforms
phosphorylate a large number of substrates including transcription
factors (e.g. c-Myc, c-Jun, NF-IL6, ATF-2, and AP-1), the
EGF receptor, phospholipase A2, tyrosine hydroxylase, protein-tyrosine
phosphatase 2C, and cytoskeletal proteins (Refs. 31, 32, and 38 and
references therein). MAPK can also phosphorylate and activate kinases,
such as p90rsk and MAPKAP kinase 2. The MAPKs themselves are
activated by phosphorylation on threonine and tyrosine residues via the
Ras/Raf/MEK/ERK pathway (reviewed in Ref. 34). Activation of MAPK
occurs during the G0/G1 transition and may be required for progression
through the cell cycle (38-42). Thus, MAPK serves to link
extracellular stimuli to cellular events involved in proliferation and
differentiation, including the cell cycle, generation of phospholipid
messengers, transcription, and translation. Other MAPKs in mammalian
cells are c-Jun N-terminal kinase/stress-activated protein kinase and p38/HOG, which are activated by stress stimuli and inflammatory cytokines.
In this study, we demonstrate that the HIV-1 Vif protein is
phosphorylated by p44/42 MAPK. Furthermore, our studies suggest that
MAPK phosphorylation of Vif plays a role in regulating HIV-1 replication and infectivity. The phosphorylation of Vif by MAPK was
demonstrated in vitro and in vivo and was shown
to occur on Thr96 and Ser165. Phosphorylation
by MAPK is likely to occur on the same sites in vitro and
in vivo based on similar patterns of two-dimensional tryptic
phosphopeptide mapping. Mutations of the Thr96 site, which
is highly conserved in HIV-1, HIV-2, and SIV, results in significant
loss of Vif function and inhibition of HIV-1 replication. These results
suggest that MAPK can regulate HIV-1 replication and infectivity by
phosphorylating Vif. This mechanism may contribute to the activation of
HIV-1 replication by mitogens and other extracellular stimuli. A
comparison of Thr96 mutations with a mutation at the
Ser144 phosphorylation site identified in a previous study
(15) suggests that Vif function and HIV-1 replication can be
significantly impaired by loss of phosphorylation at either site.
However, we cannot exclude the possibility that alternative models such
as alterations in Vif structure or the ability of Vif to interact with
a target protein may account for the impaired function of these
mutants. The biological importance of the phosphorylation site at
Ser165, which is less conserved than Thr96, or
other minor phosphorylation sites that may be utilized by MAPK (Fig. 5)
remains to be determined.
We previously identified Ser144, Thr155, and
Thr188 as major phosphorylation sites in Vif (15). These
findings, together with the identification of the Ser165
phosphorylation site, indicate that four phosphorylation sites are
located within the C-terminal region. The hydrophilic C-terminal region
of Vif is likely to be exposed on the surface of the molecule and thus
may be accessible to cellular kinase(s) (15). Moreover, the C-terminal
region appears to be important for interactions between Vif and a
membrane-associated protein (14) and possibly the p55 Gag precursor
(43). The Thr96 and Ser165 sites phosphorylated
by MAPK were not identified in our previous study, most likely because
the unstimulated cell lysates used to generate radioactive peptides for
sequencing have a relatively low level of MAPK activity. Consistent
with this possibility, two major tryptic phosphopeptides generated from
Vif phosphorylated by MAPK (Fig. 5, spots 1 and
2) were barely detectable when Vif was phosphorylated
in vitro by unstimulated cell lysates (15). Ser144, Thr155, and Thr188 are not
phosphorylated by MAPK, suggesting that other kinases also
phosphorylate Vif in vivo. Consistent with this prediction, two minor kinase bands, in addition to p44 and p42, were detected in
in-gel kinase assays using Vif as a substrate, suggesting that Vif is
also phosphorylated by other kinases present in cell lysates. Moreover,
tryptic phosphopeptide mapping of Vif phosphorylated in vivo
showed two major phosphopeptides that are not phosphorylated by MAPK
in vitro (Fig. 5, spots 6 and
7). The functional consequences of Vif phosphorylation
remain to be determined. Phosphorylation of Vif may induce a
conformational change that is important for its biological activity,
modulate its association with a membrane-associated protein (12, 14),
or promote its interaction with one of the Gag proteins (43) or a
cellular protein, thereby affecting virus assembly. Alternatively,
phosphorylation might affect the incorporation of Vif into HIV-1
virions (44, 45). However, the specificity and biological importance of
Vif incorporation into virions remain to be determined (46, 47).
An unexpected finding in this study was the demonstration that MAPK can
phosphorylate Vif at sites other than the known consensus sequences.
The optimum consensus sequence for substrate phosphorylation by MAPK is
PX(S/T)P, as determined by analysis of synthetic peptides as
MAPK substrates (35, 36). The minimum consensus required for MAPK
phosphorylation is reported to be (S/T)P, although this motif is
suboptimal compared with PX(S/T)P (31, 36). In contrast to
these studies, our data show that the sites in Vif phosphorylated by
MAPK in vitro and in vivo do not correspond to
any of the known consensus sequences. Moreover, the sequence of HIV-1
Vif does not contain any PX(S/T)P motifs. Although Vif has
several (S/T)P motifs, these sites are not the major sites of
phosphorylation by MAPK. Interestingly, synthetic peptides
corresponding to the local sequences of phosphorylation sites in Vif
were not phosphorylated by MAPK, suggesting that these sites serve as
MAPK substrates only within the context of the full-length protein.
Consistent with these findings, not all synthetic peptide substrates
corresponding to local phosphorylation sites are phosphorylated with
the same kinetics as the full-length protein (48). Thus, it is likely that a three-dimensional structure that includes Vif sequences outside
the phosphorylation site is required for recognition and phosphorylation of Vif by MAPK. Consistent with the observation that
the MAPK phosphorylation sites in Vif did not correspond to the optimum
consensus sequences, we found that the phosphorylation rate for Vif was
lower than that for MBP, one of the best MAPK substrates. In addition,
the Km for Vif phosphorylation by MAPK was
approximately 1.6 µM, which is 10-fold lower than that
for MBP (17.5 µM) (33). Therefore, the kinetic binding affinity between Vif and MAPK is likely to be high. This finding and
the demonstration that MAPK is associated with soluble cytoplasmic, membrane, and cytoskeleton fractions, a subcellular distribution pattern similar to that of Vif (12, 14, 45), raises the possibility
that Vif may directly interact with MAPK. However, we were not able to
demonstrate a direct interaction between Vif and MAPK by
co-immunoprecipitation.2
Our studies suggest that MAPK phosphorylates and activates Vif but do
not exclude the possibility that MAPK may also affect other steps of
the virus life cycle. Several steps of the HIV-1 virus life cycle
depend on cellular activation by mitogenic stimuli (16). For example,
virus replication is blocked in quiescent T cells due to incomplete
reverse transcription and lack of proviral DNA integration (49).
Stimulation with mitogens allows reverse transcription to proceed to
completion and allows subsequent virus replication to occur. Mitogenic
stimulation can also activate viral gene expression in latently
infected cells that harbor integrated proviral DNA (16). We found that
the infectivity of HIV-1 virions and subsequent virus replication is
enhanced by treatment of cells with MAPK stimulators such as serum and
PMA and inhibited by PD098059, a specific inhibitor of MAPK
activation.2 We have also shown that the HIV-1 Rev, Tat,
and p17 Gag proteins can be phosphorylated by recombinant p42 MAPK or
by immunoprecipitated MAPK in vitro.2 However,
whether these HIV-1 proteins are phosphorylated by MAPK in
vivo remains to be determined. A previous study suggested that MAPK may be associated with HIV-1 virions (50). Together, these observations raise the possibility that MAPK may also regulate HIV-1
replication by other mechanisms that are independent of Vif. For
example, MAPK may interact with and phosphorylate other HIV-1 proteins
(51), or regulate virion-associated kinases (52, 53) or cellular
kinases that associate with Tat or Nef (54-56). In addition, the
Ras-Raf pathway is activated in HIV-infected monocytes and participates
in the activation of NF- We thank J. Lee for assistance with
radioactive peptide sequencing, J. Kappes for the vaccinia virus Vif
expression system, Dr. Natalie Ahn for the pMEK-R4F plasmid, and J. Sodroski and A. Engelman for critical reading of the manuscript.
*
This work was supported by National Institutes of Health
(NIH) Grant AI36186 and by gifts from the G. Harold and Leila Y. Mathers Charitable Foundation and the Dana-Farber Friends 10. We also
acknowledge the Center for AIDS Research (NIH Grant AI28691) and Center
for Cancer Research (NIH Grant AO6514) for supporting necessary core
facilities.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported in part by AIDS Training Grant AI07387.
**
An Elizabeth Glaser Scientist supported by the Pediatric AIDS
Foundation. To whom correspondence should be addressed: Dana-Farber Cancer Institute, JF 712, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2154; Fax: 617-632-3113; E-mail:
dana_gabuzda{at}dfci.harvard.edu.
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1; MAPK, mitogen-associated protein kinase; PMA, phorbol 12-myristate 13-acetate; EGF, epidermal growth factor; MBP, myelin basic protein; PVDF, polyvinylidene difluoride; RT, reverse
transcriptase; TPCK, tosylphenylalanyl chloromethyl ketone; MEK, MAPK
kinase; ERK, extracellular signal-regulated kinase; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid
chromatography; NTA, nitrilotriacetic acid.
2
X. Yang and D. Gabuzda, unpublished observations.
Mitogen-activated Protein Kinase Phosphorylates and Regulates
the HIV-1 Vif Protein*
§¶ and
**
Department of Cancer Immunology & AIDS,
Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and the
Departments of § Pathology and Neurology, Harvard
Medical School, Boston, Massachusetts 02115
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
N3/S218E/S222D), was provided by Dr. Natalie Ahn (University of Colorado). Vif peptides were
synthesized by the Molecular Biology Core Facility at Dana-Farber. Myelin basic protein (MBP) peptides were from Santa Cruz Biotechnology. PMA, epidermal growth factor (EGF), and MBP were from Sigma.
-32P]ATP in kinase buffer). The reaction was
terminated by the addition of 2× SDS sample buffer followed by boiling
for 3 min. The reaction products were resolved by 15%
SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane
(Millipore Corp.), and detected by autoradiography.
-mercaptoethanol), and denatured in 7.5 M guanidine HCl
in buffer B (1 h at room temperature for each step). The proteins in
the gel were then allowed to renature at 4 °C overnight by extensive
washing in buffer B containing 0.04% Tween 20. Renatured kinase
activity was detected by incubating the gel for 1 h at room
temperature in a reaction buffer containing 50 mM Hepes, pH
7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 1 µM ATP, and 150 µCi
of [-32P]ATP. Unincorporated radioactivity was removed
by extensive washing with 5% trichloroacetic acid containing 1%
sodium pyrophosphate. The gel was dried, and the phosphorylated
proteins were visualized by autoradiography.
-32P]ATP, or Vif was expressed in and
isolated from 293T cells transfected with the pMEK-R4F using a vaccinia
virus expression system as described above and metabolically labeled
with [32P]orthophosphate. The samples were resolved by
electrophoresis on SDS-polyacrylamide gels and transferred to PVDF
membrane. The phosphorylated Vif band was localized by autoradiography,
excised, washed with H2O, treated with 0.5%
polyvinylpyrrolidine-360 in 0.1 M acetic acid for 30 min at
37 °C, washed with H2O again, and then digested with 15 µg of TPCK-treated trypsin (Sigma) in 50 mM
NH4HCO3, pH 8.0 at for 18 h at 37 °C.
The radioactive peptides were clarified by centrifugation, dried under
a vacuum, resuspended in pH 1.9 buffer (2.2% formic acid, 7.8% acetic
acid), and spotted onto nitrocellulose. The peptides were separated in
the first dimension by electrophoresis at pH 1.9 and in the second
dimension by thin layer chromatography in phosphochromatography buffer
(37.5% n-butanol, 25% pyridine, 7.5% acetic acid in
H2O). The radioactive peptides were detected by autoradiography.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP and visualized by autoradiography. The major
Vif kinase bands corresponded to proteins with apparent molecular
masses of 44 and 42 kDa (Fig. 1). These
molecular masses correspond to those of ERK1 and ERK2 MAPK. Kinases
with similar molecular masses were observed when in-gel kinase assays
were performed using a gel containing MBP, which is one of the best
substrates for MAPK (Fig. 1C), in addition to other bands
representing additional MBP kinases. A single band of approximately 80 kDa representing a kinase activity was observed when the
SDS-polyacrylamide gel was prepared without Vif or MBP (Fig.
1A), most likely representing a kinase with
autophosphorylating activity.
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Fig. 1.
In-gel kinase assay to detect Vif
kinase(s). CEM cell lysates were resolved under denaturing
conditions on SDS-polyacrylamide gels co-polymerized with no substrate
(A), Vif (B), or MBP (C). The proteins
in the gel were then denatured and gradually renatured, and in-gel
kinase activity was determined in the presence of
[ -32P]ATP and visualized by autoradiography.
View larger version (35K):
[in a new window]
Fig. 2.
Phosphorylation of Vif by MAPK. In
vitro kinase assays were performed using MAPK immunocomplexes
(A and B) or recombinant p42 MAPK (D).
A and B, COS-1 cells were serum-starved for
24 h followed by stimulation with serum, PMA, or EGF for 15 min at
the indicated concentrations. MAPK immunocomplexes were isolated from
the cell lysates using anti-ERK1 and anti-ERK2 and incubated with Vif
(A) or MBP (B) in kinase buffer with
[ -32P]ATP. C, detection of MAPK in
immunocomplexes by Western blotting with anti-ERK1 and anti-ERK2. MAPK
expression was not significantly affected by treatment with MAPK
stimulators. D, phosphorylation of Vif by recombinant p42
MAPK or CEM cell lysate. MAPK was incubated with 2 µg of recombinant
Vif protein for in vitro kinase assays performed in the
presence of [-32P]ATP. E, phosphoamino acid
analysis of Vif phosphorylated by recombinant MAPK. The positions of
phosphorylated Ser, Thr, and Tyr (pSer, pThr, and
pTyr) are indicated.
View larger version (16K):
[in a new window]
Fig. 3.
Kinetic and Lineweaver-Burk analysis of Vif
phosphorylation by MAPK. A, kinetic analysis of Vif
phosphorylation by recombinant p42 MAPK. Aliquots were removed at the
indicated time points, and the phosphoproteins were separated by
SDS-PAGE. The top panel shows an autoradiogram of
the gel corresponding to Vif phosphorylation at the same time points as
in the graph. The amount of 32P incorporation was
determined after SDS-PAGE by excising the gel regions containing
32P-labeled Vif and quantitation by Cerenkov counting.
B, Lineweaver-Burk analysis of the kinetics of Vif
phosphorylation by MAPK. Increasing concentrations of Vif were
incubated with recombinant p42 MAPK (50 ng) and
[ -32P]ATP. After 30 min, the reactions were terminated
and the amount of 32P incorporation was determined as in
A. The Km was determined by a double
reciprocal plot of Vif concentration versus reaction
velocity (v) expressed as pmol of 32P
incorporated into Vif.
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[in a new window]
Fig. 4.
In vivo phosphorylation of Vif by
MAPK. Vif containing a histidine tag at the N terminus was
expressed from pTM1/hVif under control of the T7 promoter using a
vaccinia virus expression system in 293T cells co-transfected with the
constitutively activated MAPK kinase expressor plasmid pMEK-R4F or the
control vector pSG5. At 36 h after transfection, cells were lysed
for detection of MAPK activity by immunoblotting with
anti-phosphorylated MAPK (A) or were labeled with
[32P]orthophosphate for 2 h and lysed with 6 M guanidine HCl, 0.1 M sodium phosphate, 20 mM imidazole, pH 8.0. Vif was purified using
Ni2+-NTA-agarose and detected by immunoblotting with rabbit
anti-Vif (B) or autoradiography (C).
D, phosphoamino acid analysis of Vif phosphorylated in
vivo in cells co-expressing pMEK-R4F. The positions of
phosphorylated Ser, Thr, and Tyr (pSer, pThr, and
pTyr) are indicated.
View larger version (61K):
[in a new window]
Fig. 5.
Two-dimensional tryptic phosphopeptide
mapping. Analysis of Vif phosphorylated in vitro by
recombinant p42 MAPK (A) or by MAPK immunoprecipitated from
HeLa cells stimulated with PMA (B) or phosphorylated
in vivo by metabolic labeling of 293T cells with
[32P]orthophosphate after infection with vaccinia virus
VV-T7 and transfection with pTM1/hVif and pMEK-R4F (D) is shown.
32P-Labeled Vif was separated by SDS-PAGE and transferred
to PVDF membrane. The 32P-labeled Vif bands were excised
and digested with TPCK-treated trypsin. Peptides were separated on
nitrocellulose plates by electrophoresis in the first dimension
followed by chromatography in the second dimension as indicated.
C, mixture of peptides from A and B.
E, mixture of peptides from A and
D.
View larger version (35K):
[in a new window]
Fig. 6.
Identification of MAPK phosphorylation sites
in Vif. A, Vif phosphorylated by recombinant MAPK
in vitro was digested by trypsin, and the peptides generated
were separated by reverse-phase HPLC under a linear acetonitrile
gradient. The arrows indicate the radioactive peptides
corresponding to those in B. B, the radioactivity
of each fraction (30 s) in A was determined by Cerenkov
counting. C, phosphoamino acid analysis of the two major
radioactive peptides. D, analysis of synthetic Vif peptides
as substrates for MAPK. Vif peptides corresponding to the sequences
surrounding the Thr96 and Ser165 sites
phosphorylated by MAPK (peptide 3, RKKRYST*QVDPEL; peptide
4, KPPLPS*VTKLRR) were incubated with MAPK in the presence
of [ -32P]ATP for 30 min at 37 °C and spotted onto
P81 phosphocellulose discs. Peptide 1 is a positive control MBP peptide
(APRT*PGGRR) that contains the optimum consensus sequence
for phosphorylation by MAPK. Peptide 2 is a negative control MBP
peptide (QKRPS*QRSKYL) that contains a protein kinase C
phosphorylation motif. Peptide 4 contains two additional arginine
residues added to the C terminus to facilitate binding to the P81
discs. Phosphorylation was quantitated by binding of 32P to
the discs after washing with 1% phosphate.
Identification of radioactive tryptic peptides of Vif by
matrix-assisted laser desorption mass spectrometry and limited
N-terminal sequencing
View larger version (14K):
[in a new window]
Fig. 7.
Analysis of T96A and T96E Vif mutants during
HIV-1 replication. The highly conserved Thr96 site in
Vif phosphorylated by MAPK or the Ser144 phosphorylation
site identified in a previous study (15) was replaced with Ala or Glu.
A, the wild-type or mutant HIV-1 proviral DNAs were
transfected into H9, CEM, or SupT1 cells, and virus replication was
monitored by quantitating RT activity in the culture supernatants at
different time points. B, virus infectivity was determined
by using virions produced by transfected H9 cells in A
normalized for equivalent amounts of RT activity (20,000 RT units) to
infect fresh H9 cells and quantitating RT activity in the newly
infected cell culture supernatants at day 11. Values shown in
A and B are the means ± S.D. from two
independent experiments. Similar results were obtained in three
independent experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B (57), a key regulator of the HIV-1 long
terminal repeat (58). MAPK is a key component of the Ras-Raf pathway
and may therefore play a direct role in the activation of NF-B in
HIV-1-infected cells. The HIV-1 virus life cycle may also be regulated
by other members of the MAPK family. For example, p38 MAPK has been
shown to activate the HIV-1 long terminal repeat (59) and appears to be
required for HIV-1 replication in T cells (60). Understanding the
signal transduction pathways that activate HIV-1 replication in
response to mitogens and extracellular stimuli may provide new insights into pathogenic mechanisms involved in AIDS and may contribute to the
development of new therapeutic strategies.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
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Abstract
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Procedures
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Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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