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(Received for publication, March 18, 1996, and in revised form, April 23, 1996)
From the Departments of Biochemistry and ABSTRACT Expression of the human immunodeficiency virus (HIV)
Nef protein has been linked to both decreased cell surface expression
of CD4 and an impairment of signal transduction. The recently reported
association of Nef with an unidentified serine kinase provides a clue
as to how Nef might exert its effects. Considering the key role of
protein kinase C (PKC) in T cell activation, we investigated the
possibility that Nef interacts with PKC. Our results, using two
approaches for detecting interactions between Nef and PKC isozymes in
Jurkat cells, show that Nef interacts preferentially with INTRODUCTION Nef is one of seven accessory proteins encoded by the human and
simian immunodeficiency viruses (HIV and SIV). The 25-kDa
nonmyristylated and the 27-kDa myristylated forms occur in the
cytoplasm, the nucleus, and the plasma, Golgi, and perinuclear
membranes (1, 2, 3, 4, 5, 6, 7). The effects of Nef on virus replication, latency, and
host cell functions and survival are unclear (8). Nef has been reported
to down-regulate the level of CD4 on the surface of infected T cells
(9, 10) and to block T cell receptor-mediated induction of
interleukin-2 in Jurkat cells, possibly via the inhibition of NF Activation of protein kinase C (PKC)1 is
required for antigen-mediated T cell activation, although the cellular
targets for its action in the signaling pathway are unknown (18, 19, 20).
Moreover, which of multiple PKC isozymes in T cells participates in the
signaling pathway has not been determined. The multiple functions
attributed to different PKC isozymes as well as the specific
intracellular localization characteristic of individual isozymes
suggests that endogenous anchoring proteins or receptors for activated
C-kinase (RACKs) exist for each isozyme (21, 22, 23). Because the
expression of MATERIALS AND METHODS The production and purification of the GST and GST-Nef
fusion proteins in Escherichia coli followed the
manufacturer's protocol (Pharmacia Biotech Inc.). The GST-Nef fusion
protein used the SR For the
Western blot analysis of PKC isozymes bound to the GST fusion proteins,
samples were extracted using glutathione beads prelabeled with GST or
GST-Nef fusion proteins as described above. SDS-polyacrylamide gel
electrophoresis sample buffer was added to the washed beads to extract
the bound proteins for Western analysis as described previously (29).
Extract from 1 × 106 cells was also run along with
the purified proteins for comparison. The nitrocellulose membranes were
probed with antibodies for Immunoprecipitations were performed on the 0.1% Triton
X-100 extracts of approximately 5 × 107 Jurkat cells
that had or had not been exposed to PMA and PHA for 5 min. Cells were
stimulated with 100 ng/ml PMA (Sigma) and 2 µg/ml
PHA (Sigma). The Nef-1-expressing cell line (133) and
the J25 parent cell line were used for the immunoprecipitations.
Construction of cell lines and their culture are described in Luria
et al. (11). Following stimulation, the cell extracts were
prepared as above, except for the addition of 0.1% Triton. 6-8 µl
of antibodies to Nef (11), To determine the translocation of
PKC isozymes (30, 31), equal amounts of protein (as determined by
Bradford assays; Bio-Rad) from the cytosolic and Triton soluble
particulate fractions of unstimulated and stimulated cells were loaded
onto 10% SDS-polyacrylamide gels for Western analysis for RESULTS AND DISCUSSION To test for an interaction between Nef and PKC, Nef was expressed
as a GST fusion protein in E. coli, purified by adsorption
on glutathione-agarose beads, and incubated with the cytosolic fraction
of Jurkat cells. PKC associated with the GST-Nef fusion protein was
assayed by measuring the phosphorylation of a specific PKC substrate
peptide (Fig. 1). PKC activity was readily detected in the
proteins extracted with GST-Nef but not with GST. Phosphorylation
occurred only in the presence of PKC activators and was inhibited by a
specific PKC inhibitor, the PKC pseudosubstrate peptide (28). Maximal
phosphorylation activity occurred in the presence of
phosphatidylserine, diacylglycerol, and EGTA, suggesting that a
calcium-independent PKC isozyme binds Nef. A decrease in kinase
activity in the presence of calcium was not observed in all experiments
and may be the result of increased degradation of the enzyme due to the
calcium in some of the experiments.
The PKC family consists of at least eleven isozymes (33), five of which
(
We have noted that under our conditions only a fraction of the
cytosolic Co-incubation of PKC with phosphatidylserine, diacylglycerol, and some
isozymes with calcium as well results in a conformational change in the
protein that allows for substrate phosphorylation and binding to RACKs
or other PKC binding proteins (23, 28). Therefore, we sought to
determine if
Another approach to determine the association of Nef with
Treatment of the Jurkat cell extracts with various preparations of
antibodies to In vivo activation of PKC generally results in the
translocation of the isozymes from the cytosol to the particulate
fraction with no net change in the total amount of the enzymes (30,
31). However, in Jurkat cells expressing Nef, the total amount of
The loss of FOOTNOTES REFERENCES
Volume 271, Number 28,
Issue of July 12, 1996
pp. 16753-16757
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
and
Molecular
Pharmacology, Beckman Center and Stanford University School of
Medicine, Stanford, California 94305
PKC. The
interaction of Nef and PKC is independent of calcium,
enhanced by phospholipid activators of PKC and not affected by a
PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and
phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to
produce the usual translocation of PKC from the cytosol to the
particulate fraction; translocation of 
PKC and 
PKC was
unaffected. Indeed, there appears to be a net loss of PKC in
Nef-expressing cells following stimulation. The loss of PKC, which
may be a result of inhibition of its binding to RACKs due to Nef
binding, could contribute to the various impairments of T cell function
associated with HIV infection and Nef expression.
B
and AP1 activation (11, 12, 13). Several reports suggest that Nef may
impair signal transduction by associating with a serine/threonine
kinase (14, 15) or disrupt calcium mobilization (16, 17) or inhibit
protein tyrosine phosphorylation and the subsequent activation of
transcription factors (17).
PKC is limited primarily to lymphocytes and skeletal
muscle and PKC is among the most abundant of the PKC isozymes in
lymphocytes (24, 25, 26), this isozyme is a plausible candidate for
involvement in the T cell receptor signaling pathway. However, the
intracellular substrates and RACKs that PKC interacts with have yet
to be determined. In this work, we have explored the possibility that
Nef interacts with PKC and more specifically with PKC. Our results
suggest that Nef may interfere with the interaction of PKC with its
endogenous anchoring proteins and that this inhibition may result in
the net loss of the isozyme.
nef-1 plasmid fused to the pGEX GST gene.
Approximately 30 µg of GST or GST-Nef fusion protein were bound to 30 µl of glutathione-agarose beads in phosphate-buffered saline
solution. The labeled beads were then incubated with the cytosolic
extract from approximately 5 × 106 Jurkat cells with
phospholipids (50 µg/ml phosphatidylserine and 0.8 µg/ml
diacylglycerol; Avanti Polar Lipids) and 1 mM calcium for
30 min at 4 °C. The beads were then washed three times with cold
phosphate-buffered saline. The Jurkat cell extract was prepared by
spinning down J25 cells (11), washing with cold phosphate-buffered
saline, and resuspended in homogenization buffer (20 mM
Tris-HCl, 1 mM EGTA, 1 mM EDTA, 5 µg/ml
soybean trypsin inhibitor, 5 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, pH 7.5). The cells were then sonicated and
centrifuged at 14,000 × g and 4 °C for 10 min. The
supernatant was then precleared with 30 µl of GST-labeled glutathione
beads for 30 min at 4 °C. To detect kinase activity, the washed
beads were used in a PKC kinase assay similar to that previously
described (27), except that the reaction was carried out at room
temperature for 5 min, and the mixture was spotted onto P81
phosphocellulose squares, which were then washed with water and
methanol before counting. In the assay, the labeled beads were
incubated with a solution containing 20 mM Tris-HCl (pH
7.5), 20 mM MgCl2, 20 µM ATP, 12 mM -mercaptoethanol, 5 µCi/ml
[
-32P]ATP, 15 µM substrate peptide, plus
or minus 50 µg/ml phosphotidylserine and 0.8 µg/ml diacylglycerol,
plus or minus 20 mM CaCl2 or EGTA, and plus or
minus 50 µg/ml pseudosubstrate peptide (28). A peptide based upon the
PKC pseudosubstrate sequence (25) with the alanine replaced with a
serine was used as the substrate (Protein and Nucleic Acid Facility,
Stanford).
, , , 
, and PKC isozymes (the
and monoclonal antibodies were obtained from Seikagaku America;
the and polyclonal antibodies were obtained from Life
Technologies, Inc./BRL; and the monoclonal antibody was obtained
from Transduction Laboratories). The monoclonal antibodies were used at
a 1:1000 dilution, whereas the polyclonal antibodies were diluted
1:300. Previous experiments found that these were the most abundant PKC
isozymes in these cells. The Western blots were developed using
chemiluminescence (ECL; Amersham Corp.).
PKC (a generous gift from Isakov and
Altman (19)) or rabbit preimmune serum were incubated with the extracts
for 2 h. The antibody-antigen complex was then precipitated with
protein A-Sepharose (Pharmacia). Alternatively, the antibodies were
biotinylated with SS-NHS-Biotin (Pierce) in order to prevent the
immunoglobulin from disassociating with the other proteins in the
extraction step. The antibody-antigen-biotin complex was then
precipitated with avidin coupled to agarose (Pierce). The antigen
complex was extracted with SDS-polyacrylamide gel electrophoresis
sample buffer. Precipitated proteins were detected by Western
analysis.
, ,
, and PKC isozymes. The cellular fractions were produced from
cell homogenates by a 30-min 100,000 × g
centrifugation followed by a 0.1% Triton extraction of the pellet and
recentrifugation. The supernatant from the final centrifugation was
used as the Triton soluble particulate fraction. The larger molecular
weight protein detected by the antibody to PKC, which translocates
with stimulation, may be or PKC as reported by others (32). The
Triton soluble particulate fraction does not contain the nuclei or
other cellular components not extracted with 0.1% Triton. Western
analysis of the Triton nonextracted pellet from unstimulated or
stimulated cells revealed little PKC remaining in the pellet
(results not shown).
Fig. 1.
A kinase activity from Jurkat cell extracts
with characteristics of PKC bound to a GST-Nef fusion protein. The
kinase assay was performed using glutathione beads that had been
prelabeled with the fusion proteins and used to purify binding proteins
from Jurkat cell cytosolic extracts. The kinase activity was determined
in the presence of phospholipids (phosphotidylserine and
diacylglycerol, PS/DAG) and calcium (Ca),
phospholipids and EGTA, EGTA alone, or phospholipids, calcium, and the
PKC pseudosubstrate inhibitory peptide. A peptide based upon the PKC
pseudosubstrate sequence (25) with the alanine replaced with a serine
was used as the substrate. The PKC pseudosubstrate inhibitory peptide
is a highly specific inhibitor of PKC that is not isozyme-specific. The
results are expressed as the averages of duplicate assays and are from
one of three experiments with similar results.
, , , , and ) were clearly detected by Western blot
analysis in Jurkat cells (Fig. 2A,
cytosol). To identify which PKC isozyme bound to Nef, the
purified GST-Nef and associated proteins recovered after incubation
with Jurkat cell cytosol were electrophoresed, and the protein blots
were analyzed with antisera specific for the five PKC isozymes (Fig.
2A). Of the five PKC isozymes detected in Jurkat cells, only
PKC bound to the GST-Nef fusion protein; lower amounts of the PKC
isozymes bound to GST alone due to nonspecific interactions. Further
evidence supporting our conclusion that PKC binds Nef is the
observation that the addition of increasing amounts of purified,
unbound Nef proportionally lowered the binding of PKC to the fusion
protein (Fig. 2B). In addition, treatment of the GST-Nef
fusion protein with thrombin, which cleaves Nef from GST, eliminated
the binding of PKC to the glutathione beads (results not shown).
Fig. 2.
Western blot analysis of PKC isozymes from
Jurkat cell cytosolic extracts that bind to a GST-Nef fusion
protein. Glutathione beads were incubated with GST alone or with
the GST-Nef fusion protein and were used to purify binding proteins
from Jurkat cell extracts as described (A). For comparison,
20% of the cytosol prepared from the Jurkat cells was run in parallel
on the Western blot (cytosol) for each isozyme probed (approximate
molecular masses: PKC, 82 kDa; PKC, 82 kDa; PKC, 80 kDa;
PKC, 90 kDa; PKC, 69 kDa). The results are from one of four
independent experiments with identical results. Preincubation of the
Jurkat cell extract with increasing amounts of Nef, purified from
E. coli (11), decreased the binding of PKC to the GST-Nef
bound to the beads (B; approximate molecular mass of
detected protein is 80 kDa). For comparison, approximately 30 µg of
GST-Nef fusion protein was bound to the beads as determined by Western
blot analysis of extracted proteins.
PKC (about 5%) is bound to the GST-Nef, even though the
amount of GST-Nef is in molar excess over the amount of PKC. In
addition, PKC activators phosphatidylserine and diacylglycerol were
present in sufficient concentrations to fully activate the PKC.
Moreover, when the cytosolic fraction, which had already been reacted
with GST-Nef, was reincubated with fresh GST-Nef, there was no
additional binding of PKC. This suggests that PKC in the
cytosolic fraction is heterogeneous with respect to its ability to bind
Nef.
PKC needs to be activated to bind Nef. To this end
GST-Nef, immobilized on glutathione beads, was incubated with Jurkat
cell cytosol in the presence or the absence of PKC activators. The
recovered binding proteins were analyzed by Western blot analysis (Fig.
3). Binding of PKC was maximal in the presence of
phosphatidylserine, diacylglycerol, and EGTA, consistent with the
results obtained with the PKC kinase assay (Fig. 1). Nef has been
reported to be a PKC substrate (34). However, the interaction between
PKC and Nef was not via the PKC substrate binding site. Similar to
PKC binding to other proteins that have been shown to interact with PKC
such as RACKs (21, 22), a PKC peudosubstrate peptide did not inhibit
Nef binding to PKC (Fig. 3).
Fig. 3.
PKC binding to the GST-Nef fusion protein
requires phospholipids and is not inhibited by a PKC pseudosubstrate
peptide. GST or GST-Nef fusion proteins immobilized onto
glutathione beads were incubated with Jurkat cell cytosol in the
presence of phosphotidylserine and diacylglycerol (PS/DAG),
calcium (Ca), EGTA, or the PKC pseudosubstrate peptide.
PKC binding was determined as above (approximate molecular mass of
detected protein is 80 kDa).
PKC relied
on co-immunoprecipitation. Antibodies directed against Nef
immunoprecipitated both Nef and PKC from extracts of Jurkat cells
that express Nef (Fig. 4A). By contrast, PKC
was not co-precipitated with anti-Nef antibodies (results not shown),
nor did preimmune serum or protein A-Sepharose beads alone precipitate
Nef or PKC (Fig. 4A). PMA and PHA stimulation of the
Nef-expressing cells did not affect the amount of PKC that
co-precipitated with Nef. Considering that the cells are grown in media
containing growth factors, it is possible that there is a sufficient
amount of activated PKC to associate with the endogenously produced
Nef; such a possibility is inferred from the observation that some
particulate fraction-associated PKC is usually present in
Nef-expressing Jurkat cells even before stimulation.
Fig. 4.
PKC and Nef co-immunoprecipitate from
Jurkat cells expressing Nef. Immunoprecipitations were performed
using extracts of unstimulated or stimulated (PMA and PHA) cells
expressing Nef or control cells not expressing Nef, using polyclonal
antibodies against Nef, PKC, or rabbit preimmune serum or protein
A-Sepharose beads alone (A). For control precipitations,
equal volumes of unstimulated and stimulated cells were pooled
(pool) or extracts from an equal number of J25 Jurkat cells
not expressing Nef were used. Alternatively, Nef and PKC were
co-precipitated using biotinylated antibodies to PKC (B).
Biotinylated antibodies to Nef and biotinylated rabbit preimmune serum
were also used. Precipitated proteins were detected by Western analysis
with antibodies against PKC and Nef (approximate molecular masses:
PKC, 80 kDa; Nef, 27 kDa).
PKC immunoprecipitated PKC but failed to
co-precipitate detectable levels of Nef using protein A-Sepharose (Fig.
4A). However, Nef could be co-immunoprecipitated with PKC
using biotinylated antibodies to PKC and avidin-agarose (Fig.
4B). Using biotinylated antibodies removed the background
signal resulting from the immunoglobulin light chain that migrated at
the same molecular weight as Nef on SDS-polyacrylamide gel
electrophoresis. This method greatly improved the sensitivity of the
Western for immunoprecipitated Nef but also lowered the amount of
antigen that was precipitated. In particular, biotinylated antibodies
to Nef did not consistently co-precipitate PKC (Fig. 4B),
possibly because of interference by the biotin on the binding of the
antibodies to the complex.
PKC declined after 5 min of PMA and PHA stimulation (Fig.
5A); by comparison the total level of PKC
remained unchanged after stimulation. To examine this isozyme-specific
difference further, the translocation of four PKC isozymes was compared
in PMA- and PHA-activated control and Nef-expressing Jurkat cells (Fig.
5B). Translocation of , , and PKC isozymes was
observed after 5 min of PMA and PHA stimulation in the control Jurkat
cells; translocation of the and isozymes was virtually
unchanged in the Jurkat cells expressing Nef (PKC localization is
unaffected by PMA, whereas the higher molecular weight cross-reactive
proteins may be other PKC isozymes (32)). However, PKC translocation
was disrupted in the cells expressing Nef; stimulation resulted in the
loss of PKC from the cytosolic fraction without a concomitant
increase in the level of PKC in the particulate fraction.
Furthermore, as pointed out earlier, even without stimulation, there
was a greater proportion of PKC in the particulate fraction of
Nef-expressing cells than in control cells (Fig. 5B).
Fig. 5.
Nef expression in Jurkat cells correlates
with the specific loss of PKC following PMA and PHA
stimulation. The total level of PKC declined in Nef-expressing
cells following PMA and PHA stimulation, whereas total PKC levels
remain unchanged (A; approximate molecular masses: PKC,
80 kDa; PKC, 82 kDa; Nef, 27 kDa). Triton extracts of total cell
homogenates were prepared of the stimulated and unstimulated
Nef-expressing cells and subjected to Western analysis. The levels of
Nef in the two cell extracts were also determined to demonstrate that
equal amounts of protein were loaded in both lanes. Control Jurkat
cells or Nef-expressing cells were stimulated with PMA and PHA and
fractionated into cytosol and Triton-soluble particulate fractions to
further analyze the loss of PKC (B). Samples containing
equal amounts of protein were analyzed by Western blot to determine the
relative levels of , , , and PKC before and after
stimulation in the two cellular fractions (approximate molecular
masses: PKC, 80 kDa; PKC, 82 kDa; PKC, 90 kDa; PKC, 69 kDa,
lower band). The results are from one of six independent
experiments with similar results.
PKC following PMA and PHA treatment of Nef-expressing
cells may be a result of the isozyme's interaction with Nef. This
could result from the failure of PKC to bind to its endogenous
anchoring protein(s) or RACKs (21, 22, 23) following activation of the PKC.
Activated PKC has been shown to be more sensitive to proteolysis (36).
Inhibition of binding of activated PKC to its endogenous binding
proteins or RACKs may leave the PKC more susceptible to degradation.
Indeed, an inhibition of translocation and a net loss of PKC following
activation was observed in oocytes in which translocation inhibitors
(either a purified RACK protein or peptides based upon sites of
interaction between PKC and RACKs) were introduced by microinjection
(35, 37, 38). Nef may act as an inhibitor of PKC translocation in
lymphocytes in a similar manner. Quite possibly, the loss of PKC or
its inappropriate binding to its targets could account for the various
phenotypic impairments of T cell function associated with Nef and HIV
infection. Further work is required to determine the molecular basis
for the loss of PKC in cells expressing Nef and how this disruption
in signal transduction affects T cell activation.
§
To whom correspondence should be addressed. Tel.: 415-723-6170;
Fax: 415-725-4951.
1
The abbreviations used are: PKC, protein kinase
C; RACK, receptor for activated C-kinase; GST, glutathione
S-transferase; PMA, phorbol 12-myristate 13-acetate; PHA,
phytohemagglutinin.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Datta, H. Kojima, K. Yoshida, and D. Kufe Caspase-3-mediated Cleavage of Protein Kinase C theta in Induction of Apoptosis J. Biol. Chem., August 15, 1997; 272(33): 20317 - 20320. [Abstract] [Full Text] [PDF]
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K. Coates, S. J. Cooke, D. A. Mann, and M. P. G. Harris Protein Kinase C-mediated Phosphorylation of HIV-I Nef in Human Cell Lines J. Biol. Chem., May 9, 1997; 272(19): 12289 - 12294. [Abstract] [Full Text] [PDF]
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Y. Izumi, S.-i. Hirai, Y. Tamai, A. Fujise-Matsuoka, Y. Nishimura, and S. Ohno A Protein Kinase Cdelta -binding Protein SRBC Whose Expression Is Induced by Serum Starvation J. Biol. Chem., March 14, 1997; 272(11): 7381 - 7389. [Abstract] [Full Text] [PDF]
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