(Received for publication, July 13, 1995; and in revised form, November 3, 1995)
From the
Infection with Herpesvirus saimiri, a T lymphotropic
virus of non-human primates, immortalizes human T cells in
vitro. The cells show a mature activated phenotype and retain
their antigen specificity. We have previously shown that in H.
saimiri transformed cells a viral gene product termed tyrosine
kinase interacting protein (Tip) associates with the T cell-specific
tyrosine kinase p56 and becomes phosphorylated
by the enzyme on tyrosine residues. Here we show that p56
is activated by recombinant and native Tip in cell-free
systems. A dramatic increase of Lck activity was also observed in T
cell lines transfected with Tip. p60
and
p53/56
, the other Src-related kinases expressed
in H. saimiri transformed T cells, did not phosphorylate Tip,
and they were not activated by the protein. The selective activation of
p56
by Tip could contribute to the transformed
phenotype of H. saimiri infected cells, and it might explain
the T cell selectivity of the transformation event.
Certain strains of Herpesvirus saimiri readily
transform human T cells to continuous growth in cell
culture(1) . Remarkably, the antigen-specific response of the
immortalized T cells as well as their mature and activated phenotype
are very stable. The T cell receptor-CD3 complex, the CD4 or CD8
coreceptors, CD2, and the IL-2 ()receptors remain present
and functionally competent over many months in
culture(2, 3, 4, 5, 6) ,
suggesting that the signaling apparatus of mature T cells may be
required for the viral transformation. The viral genome harbors
approximately 75 open reading frames. Many have been tested for
transcription in transformed human cells but, so far, only ORF1 and
ORF2 (7) encoded on a single bicistronic mRNA have been found
in the permanently growing cells. We have previously shown, that the
gene product of ORF1, tyrosine kinase interacting protein (Tip) is
expressed in transformed cells. Tip associates with the protein
tyrosine kinase p56
and can be phosphorylated by
this enzyme in vitro(8) . p56
is a non-receptor tyrosine kinase of the Src family which is
selectively expressed in thymocytes and mature T cells. The kinase
tightly associates with the coreceptor molecules CD4 or CD8 and becomes
activated after cross-linking of these receptors on the cell surface.
One of the substrates of p56
is the
-chain
of the T cell receptor, and T cells deficient in p56
fail to respond to T cell receptor mediated signals. This
attributes a crucial role to the enzyme in the antigen-specific
response of T cells(9, 10) . The ORF2-encoded saimiri
transformation-associated protein (StpC) is also expressed in H.
saimiri transformed human T cells and might complement the action
of Tip. StpC is a strong oncoprotein in rodent fibroblasts and
epithelial cells of transgenic mice, but not in T
cells(11, 12) . The mechanism of oncogenesis by StpC
is not known. Transformation of T cells probably requires the action of
Tip on the T cell-specific kinase p56
in
addition to StpC. Here we further characterize this interaction and
show that Tip selectively interacts with p56
,
but not with the other Src-related kinases p60
or p53/56
which are expressed in
human T cells immortalized by H. saimiri. Furthermore, this
interaction leads to a dramatic activation of the enzyme. This may
explain why many human cell types can be infected by H. saimiri but only T cells are transformed to permanent growth.
Figure 4:
Enzymatic activation of p56 by recombinant and native Tip. p56
was
precipitated from Jurkat cells (200 µg of protein/sample), and an
immune complex kinase assay was carried out in the presence of 2 µg
of enolase as exogenous substrate. Increasing amounts of
-Gal-Tip (A),
-Gal (B), and Lck-depleted BW
-Tip
lysate as a source of native Tip (C) were added. The
Lck-depleted lysate of untransfected BW
cells served as
control in C. In D, increasing amounts of
-Gal-Tip were added to 0.5 µg of recombinant
p56
, and a phosphotransferase reaction was
performed.
-Gal was used as control. The proteins were separated
by 8% SDS-PAGE, and the gels were dried and exposed to x-ray film. The
positions of Lck, Tip,
-Gal-Tip, and
-Gal are on the right, molecular mass standards are on the left.
Figure 6:
Tyrosine phosphorylation analysis. A, transformation by H. saimiri increases the
cellular content of phosphotyrosine. Phosphotyrosine-containing
proteins were precipitated from 2 mg of protein of T cell lines
transformed by H. saimiri, from their nontransformed parental
clones and from phytohemagglutinin-stimulated T cell blasts using the
monoclonal antibody PY-20. For control, only PY-20 but no cell lysate
was added. After separation by 12% SDS-PAGE and transfer to a
nitrocellulose membrane, bands containing phosphotyrosine were revealed
with a polyclonal antiserum and I-protein A followed by
autoradiography. B, Tip is phosphorylated on tyrosine residues in vivo. Phosphotyrosine-containing proteins were precipitated
from cell lysates (200 µg of protein) as described above (lanes
1-3) and separated by 8% SDS-PAGE. 50 µg of whole cell
lysate from the same cell lines was loaded in lanes 4-6 of the same gel. Proteins were transferred to nitrocellulose, and
the blot was probed with the anti-Tip monoclonal antibody BNI2. Bands
were visualized by enhanced chemiluminescence. Tip expression in H.
saimiri transformed cells (e.g. 39-HVS) regularly escaped
detection by immunoblotting. The positions of Tip and the Ig heavy and
light chains are shown on the right, the molecular mass
markers are on the left.
A fusion protein of amino-terminally
truncated Tip and -galactosidase as well as
-galactosidase
alone were gifts of B. Biesinger, Erlangen, Germany, and have been
described(8) . Tip-encoding sequences on the plasmid p488PX
(gift of B. Biesinger(7) , EMBL accession number M55264) were
amplified by polymerase chain reaction with the following primers:
forward, CGCGGGCTCGAGATGGCAAATGAAGGAGAAGAA; reverse,
CCGGCCGCGGCCGCTTAACTTTTCATTCCTATATG. This inserted a XhoI
restriction site 5` and a NotI restriction site 3` of the
coding regions which were used to clone the gene into the eukaryotic
expression vector BCMGSNeo (gift of H. Karasuyama(17) ).
Plasmids were transfected into BW
cells by electroporation.
One day after transfection, 1 mg/ml G418 was added to the cultures for
selection. Growing cell lines were characterized by immunoprecipitation
of p56
followed by phosphotransferase assay and
reprecipitation with the anti-Tip serum. A cell line expressing large
amounts of Tip was cloned to obtain BW
-Tip.
Figure 1:
Expression of p53/56 in
addition to p56
and p60
in human T cells
after infection with H. saimiri. Lysates were prepared from
the nontransformed T cell clone 61/39 (A) and from the same T
cell clone 6 months after transformation by H. saimiri (39-HVS, B). Src-related kinases were immunoprecipitated
from 500 µg of total cell protein/sample except for p56
where 100 µg of total cell protein was used. S.
aureus particles without anti-kinase serum served as control. An in vitro kinase assay was performed, and the resulting
phosphoproteins were separated by 8% SDS-PAGE. The two gels were
electrophoresed in parallel, dried, and exposed to x-ray film for 2 h
to reveal autophosphorylation. Molecular mass standards are on the right.
Figure 2:
p56 but not
p60
or p53/56
specifically phosphorylates
recombinant Tip in vitro. p56
was
immunoprecipitated from Jurkat cell lysates (600 µg/sample),
p60
from Lyn-depleted lysates of BJAB cells (300
µg/sample), and p53/56
from lysates of Raji
cells (900 µg/sample). S. aureus particles without
anti-kinase antibody served as control. An immune complex kinase assay
was performed without addition or in the presence of 1 µg of either
the
-Gal-Tip fusion protein or of unfused
-Gal as indicated.
Molecular mass standards are shown on the left, and the
positions of
-Gal-Tip or
-Gal are on the right.
Figure 3:
Characterization of the murine T cell line
BW transfected with p40
. A,
association of p40
with Lck. p56
was precipitated from 100 µg of protein of the
transfected T cell line BW
-Tip (+) and from 3000 µg
of protein of its mock-transfected counterpart
BW
-neo(-). An immune complex kinase assay was performed
and proteins were separated by 8% SDS-PAGE. The gel was dried and
autoradiographed. Different amounts of total cellular protein were used
for immunoprecipitation in order to achieve comparable levels of Lck
autophosphorylation (lanes 1 and 2). This was
necessary in order to demonstrate convincingly the absence of a 40-kDa
band in the BW
-neo cells. The 40-kDa band in Lck
immunoprecipitates from BW
-Tip was then identified by
dissociation of the immune complexes and reprecipitation of Tip with a
specific antiserum (lanes 3 and 4). Proteins in these
precipitates were separated on the same gel which was dried and
autoradiographed. B, expression of p56
.
Lck was immunoprecipitated from different protein amounts of
BW
-Tip and of Jurkat cells as indicated and subjected to 8%
SDS-PAGE. After transfer to a nitrocellulose membrane, the Lck protein
amount was visualized by probing with a rabbit serum directed against
Lck followed by
I-protein A. The immunoblot was then
autoradiographed. PhosphorImager analysis revealed that
BW
-Tip contained 220-fold less Lck than Jurkat cells. The
positions of Lck and Tip are indicated on the left, molecular
mass standards are on the right.
Figure 5:
Expression of Tip increases the enzymatic
activity p56 in the murine T cell line BW
.
p56
was precipitated from different amounts of
cell lysate from the Tip-transfected T cell line BW
-Tip and
from its mock-transfected counterpart BW
-neo. For
determination of enzymatic activity, immunoprecipitates were subjected
to an immune complex kinase assay, and the resulting proteins were
separated by 8% SDS-PAGE (A). For measurement of protein
abundance, immunoprecipitates were separated on 8% SDS-PAGE,
transferred to nitrocellulose membranes, and probed with serum directed
against Lck followed by
I-protein A (B). The
dried gel (A) and the immunoblot (B) were then
autoradiographed. Controls without precipitating antibody are boxed. Note the large amounts of cell lysate necessary to
visualize Lck from these murine cells on immunoblots. The specific
activity of Lck in BW
-Tip is, therefore, much higher than
what is usually observed in T cells. The positions of Lck, enolase, and
Tip are shown on the right, molecular mass standards are on
the left.
Previously we have suggested that two factors cooperate in
the transformation of human T cells by H. saimiri with StpC
acting as the basic oncoprotein complemented by the T cell-specific
action of Tip(8) . The results of our analysis of the Tip/Lck
interaction presented here lend support to our hypothesis. Tip binds to
and directly activates the Src-related kinase p56, which
is expressed in large amounts in T cells and thymocytes. Because of
substrate competition at high concentrations of Tip, the maximal
enzymatic activation of Lck could not be determined. The factor of
3-6 observed in our system is, therefore, a conservative
estimate. Besides Lck, which is by far the most abundant Src-related
kinase in human T cells, we observed significant activities of
p60
and, unexpectedly, also of p53/56
in
transformed T cells. However, neither p60
nor
p53/56
was affected by Tip.
Expression of
p53/56 in T cells is unusual. It has been observed
previously only after infection of T cells by the retrovirus
HTLV-I(18) . This could be partially explained by trans-activation of the Lyn promoter by the HTLV-I encoded
transcription factor p40
. However, p40
is
not related to the H. saimiri encoded
p40
(19) . Furthermore, the appearance of Lyn in
HTLV-I-infected T cells was accompanied by a gradual loss of Lck
activity, which correlated with dedifferentiation of these cells to
IL-2-independent growth (18) . (
)Neither decrease of
Lck abundance and activity nor IL-2 independence was observed in human
T cells transformed to permanent growth by H. saimiri. The
mechanisms of the aberrant activation of Lyn in these cells are not
known at present.
What might be the consequences of the Tip-mediated
activation of Lck, an enzyme which plays a pivotal role in T cell
signaling? It is well documented that p56 which is
constitutively active by virtue of mutation or deletion of its
regulatory tyrosine 505 acts as an oncoprotein in fibroblasts. In these
cells, wild-type p56
was not effective even when
overexpressed(20, 21, 22) . In thymocytes of
p56
transgenic mice, increased expression of both
wild-type and active mutants of Lck induced the formation of
thymomas(23) . Therefore, activation of p56
by
Tip may contribute to the transformed phenotype of H. saimiri infected T cells. On the other hand, transfection of Tip into
fibroblasts did not result in transformation(11) . However,
fibroblasts do not express Lck, and our data show that enzymatic
activation by Tip is highly selective for this Src family member. In
agreement with this, no kinase activity was associated with Tip in
transfected murine fibroblasts. (
)
Transfection with
constitutively active p56 resulted in increased
responsiveness to T cell receptor-mediated signals in a murine T cell
hybridoma, a model for mature T cells(24) . Enhanced tyrosine
phosphorylation in response to TcR triggering in T cells transformed by H. saimiri has been documented earlier(3) . To
investigate whether this could be the result of Lck activation by Tip,
we first attempted to demonstrate an increase of the specific enzymatic
activity of Lck in T cells transformed by the virus. While a dramatic
increase of p56
activity was readily observed in T cells
overexpressing Tip after transfection, we could not clearly show it in
the T cell lines transformed by wild-type virus. This may be due to the
very low level of Tip expression in these cells. Activation of a small
fraction of Lck by the associated Tip would then be masked by the
excess of nonactivated enzyme. However, the small fraction of
Tip-activated Lck might be non-randomly distributed in the cells and
still have a significant effect on the phosphorylation of selected
substrates. We have, therefore, tested the basal level of tyrosine
phosphorylation in T cells without any further stimulation. This most
clearly reflects the influence of H. saimiri. Three bands with
molecular masses of around 70, 55, and 30 kDa were more strongly
phosphorylated in the virally transformed T cells. The prominent 55-kDa
band, which faintly also appears in the nontransformed T cells, very
likely corresponds to p56
, which is by far the most
abundant and most active Src family kinase in all our T cell lines. Lyn
is much less active than Lck in H. saimiri transformed T
cells, and it is not expressed in the nontransformed parental cell
lines. Increased phosphorylation of p56
is probably a
direct effect of its activation by Tip. However, it cannot be excluded
that p56
or one of the other prominent bands on the
phosphotyrosine immunoblot might also be a substrate of
p53/56
. Other nonidentified kinases or phosphatases may
also play a role.
One of the most striking changes in the behavior
of human T cells transformed by H. saimiri is their
hyper-reactivity to ligation of CD2. Because the ligand for CD2, LFA-3,
is also expressed on these cells, cell contact leads to ligation of
CD2, which results in autostimulation, IL-2 production, and autocrine
growth(6) . Blockade of the CD2/LFA-3 interaction can halt the
growth of H. saimiri transformed T cells, so that this
autocrine loop appears to be essential for the transformed phenotype (6) . ()p56
has been found in complex
with CD2, and the enzyme becomes activated after CD2
cross-linking(25, 26) . Therefore, activation of
p56
by Tip could enhance the T cell responses to CD2.
However, the dissection of CD2-mediated signaling in H. saimiri transformed T cells requires further investigation.
The
mechanism by which Tip activates p56 is not understood.
It is likely that the hydrophobic stretch at the carboxyl terminus of
Tip inserts into the cell membrane and brings the molecule into
proximity of Lck. In fact, the Tip homologue of a related H.
saimiri strain is located in the outer cell membrane(27) .
The Tip sequence contains a 10-amino acid stretch with strong
similarity to a sequence in the carboxyl terminus of Src family kinases
as well as a type II SH3-domain binding motif(8) . These are
necessary and sufficient for efficient binding of Tip to
Lck(28) . Analysis of the crystal structure of the regulatory
domains of Lck revealed dimerization of the SH2/SH3 domain structure
and binding of the regulatory carboxyl-terminal peptide containing
tyrosine 505 to the contact area of the two Lck molecules(29) .
At the cell membrane, such a closed conformation could block the
catalytic domains of Lck and inactivate the enzyme. The authors suggest
that ligand binding to the SH2 or SH3 domain of Lck might induce the
open, enzymatically active state(29) . We have shown that Tip
is phosphorylated on tyrosine residues in transfected T cells, and that
it can be phosphorylated by Lck on tyrosine residues in cell-free
systems(8) . But phosphotyrosine does not interfere with the
association of Tip with Lck making involvement of the SH2 domain of Lck
unlikely. However, interaction of the class II SH3 binding motif with
the SH3 domain of Lck could induce the active open conformation of the
enzyme.
Polyoma virus middle T antigen is another viral protein
which binds and activates Src family kinases, namely
p60, p62
, and
p59
(30) . Besides Src family kinases, middle T
binds a multitude of cellular proteins involved in signal transduction,
the function of which seems to converge in the activation of the
mitogen-activated protein kinase pathway(30) . Whether there
are other signaling molecules associated with Tip remains to be
discovered. It could be that the oncoprotein StpC complements Tip so
that the two factors together achieve a function comparable to that of
polyoma middle T antigen.
The relevance of Tip-induced activation of
p56 for the transformation of human T cells by H.
saimiri remains to be finally proven. Our data support the notion
that in the presence of the oncoprotein StpC the action of Tip on the T
cell kinase p56
could be a decisive factor. This would
explain the T cell selectivity of the transformation by H.
saimiri.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M55264[GenBank].