(Received for publication, May 3, 1995; and in revised form, June 27, 1995)
From the
A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.
Proliferation of mature T cells is induced by a multistep
process following exposure to antigen-presenting cells. Antigen
presentation can be mimicked by the cross-linking of the T cell
receptor and certain T cell surface molecules with specific
antibodies(1, 2) . Both modes of T cell receptor
stimulation lead to rapid tyrosine phosphorylation of cellular proteins
followed by an increase in intracellular free calcium. Phosphorylation
results from the sequential activation of several tyrosine
kinases(1, 3, 4) . A central role in T cell
activation has been assigned to the tyrosine kinase Lck. A T cell line
defective for lck expression fails to induce tyrosine
phosphorylation after stimulation(5) . As a member of the Src
kinase family, Lck consists of a short unique region, an SH3 ()and an SH2 domain followed by the catalytic domain and a
regulatory carboxyl terminus. A myristylation site at the amino
terminus attaches the protein to the membrane. The amino-terminal
unique sequences are responsible for binding to membrane-anchored
surface molecules like CD4 or CD8(6) . The SH2 and SH3 domains
bind specific substrates and downstream effectors of Lck(7) .
Herpesvirus saimiri (HVS), a member of the 2 group of
herpesviruses, naturally infects squirrel monkeys (Saimiri
sciureus) of South America. HVS persists in T lymphocytes of the
natural host without any apparent disease, but infection of other
species of New World primates results in fulminant lymphomas,
lymphosarcomas, and leukemias of T cell origin(8) . A
pronounced divergence among different strains of HSV has been localized
to the left-end of viral genomic DNA, and this has led to
classification into three subgroups, A, B, and C (9, 10) . Strains from subgroups A and C are highly
oncogenic and are able to immortalize common marmoset T lymphocytes in vitro to interleukin 2-independent
growth(11, 12) . Subgroup C strains are further
capable of immortalizing human and rhesus monkey lymphocytes into
continuously proliferating T cell lines(13) .
Nucleotide sequence analysis of the entire HVS genome revealed a number of genes with homology to cellular proteins, some of which are likely to contribute to T cell transformation(14) . These include the STP oncogene(15, 16, 17) , superantigen homolog(18) , interleukin 8 receptor homolog(19) , and virus-encoded cyclin(19, 20) . Recently, the product of the gene (orf1) adjacent to STP-C488 at the left end of the viral genome was identified in transformed T cells(21) . Orf1 did not show transforming activity in rodent fibroblast cells(17) , but the protein was found to be associated with the major T cell tyrosine kinase Lck and phosphorylated on tyrosine residues by purified Lck in several cell-free assay systems(21) . Thus, it was designated as tyrosine kinase interacting protein (Tip)(21) . However, any role for Tip in viral-induced cell growth transformation is yet to be defined.
As a first step toward analyzing the function of Tip, we have now localized the Lck-binding domains of Tip protein. Our experiments show that two structural motifs as well as the connecting sequences are necessary and sufficient for efficient Lck binding activity. These two structural elements are a proline-rich segment similar to sequences known to bind to SH3 domains and a motif homologous to the carboxyl-terminal regulatory region of Src-related tyrosine kinases. HVS has apparently acquired these structural elements in Tip for binding to cellular protein kinases.
All mutations in the tip gene were generated with polymerase chain reaction using oligonucleotide-directed mutagenesis(23) . The amplified DNA fragments containing mutations in tip were purified and cloned into pBluescript KS+ vector. Each tip mutant was completely sequenced to verify the presence of the mutation and the absence of any other changes. After confirmation of the DNA sequence, DNA containing the desired tip mutation was recloned into pFJ or pBKCMV vector for gene expression or into pGEX4T for production of bacterial GST-Tip fusion protein.
Figure 1: Schematic diagram of the structural organization of Tip and summary of in vitro binding of wild-type and mutant forms of GST-Tip to Lck. Box R is the repeat of KLSSCSEETT sequence, Y is tyrosine residue, and hydrophobic at the carboxyl terminus is the hydrophobic domain. Amino acid residues in the shadedbox of the CSKH motif represent the sequence highly homologous to the carboxyl termini of Src-related kinases. XPPLPXR is the consensus sequence for SH3 binding motif(26, 27, 28) . In the Tip mutant called mSH3B, proline residues at amino acids 175, 177, 178, 180, 181, and 183 in SH3B element were changed to alanines. Results for in vitro binding assays with various GST-Tip fusion proteins with Lck from insect cells were summarized in the bottom of figure. ++++, strong binding; -, no binding.
To identify structural elements required for complex
formation between Lck and Tip, we first established an in vitro binding system. Gene sequences for Tip lacking the
carboxyl-terminal hydrophobic region were fused to the GST gene to
produce bacterial fusion protein. Purified GST-Tip protein was mixed
with precleared S-labeled insect cell lysates containing
Lck, washed extensively, and resolved by SDS-PAGE. As shown in Fig. 1and 2A, GST-Tip fusion protein efficiently bound in vitro to Lck, while GST protein alone did not.
Additionally, the GST-Tyr
mutant, which lacks the
amino-terminal repeat sequence, still bound to Lck as well as wild-type
GST-Tip. Thus, purified GST-Tip protein bound efficiently to Lck in
vitro, and the amino-terminal repeat sequence was not required for in vitro complex formation with Lck.
Figure 2:
SH3B motif of Tip is essential but not
sufficient for binding to Lck. A, SH3B of Tip is important for
binding to Lck. B, additional motifs between
Tyr(Y) and Tyr(Y
) are required for
binding to Lck. Glutathione-Sepharose beads containing 5 µg of GST
or various GST-Tip fusion proteins were mixed with
S-labeled cell lysates containing Lck from insect cells
followed by three washing steps with lysis buffer. 5 µg of anti-Lck
antibody was used for immunoprecipitation of Lck as control (B, lane1). Associated proteins were
resolved in SDS-PAGE and autoradiographed.
Since the SH3B element is essential but not sufficient
for binding to Lck in vitro, we investigated which additional
regions of Tip may participate in binding to Lck. A series of deletion
mutants of the tip gene were generated by polymerase chain
reaction and fused to the GST fusion expression vector. As described
above, purified bacterial GST fusion proteins were mixed with
precleared S-labeled lysates from Sf9 insect cells
infected with Lck baculovirus, and associated proteins were resolved by
SDS-PAGE. GST-Tip, GST-Tyr
, GST-Tyr
, and
GST-Tyr
efficiently bound to Lck in vitro (Fig. 2B). However, the GST-Tyr
deletion mutant no longer bound to Lck (Fig. 2B).
Again, GST-SH3B did not bind to Lck (Fig. 2B). Thus,
amino acids between 127 and 155 together with the SH3B element are
required for efficient Lck binding in vitro.
The
requirement for amino acids 127-155 suggested that CSKH is likely
to be important for Lck binding. To test the importance of this CSKH
element for Lck binding, additional deletion mutants were generated and
fused into GST expression vector. GST-Tyr,
GST-Thr
and GST-Glu
were capable of binding
to Lck in vitro, while GST-Thr
/mSH3B,
GST-Glu
/mSH3B, GST-Tyr
, and GST-Pro
were deficient in binding to Lck (Fig. 3). To investigate
this further, point mutations were introduced into the CSKH element of
GST-Glu
, whose binding activity is similar to that of
wild-type GST-Tip (see Fig. 5).
GST-Glu
/Ser
Arg/Phe
His/Leu
Met contained changes of
serine to arginine, phenylalanine to histidine, and leucine to
methionine, and GST-Glu
/Phe
Cys had
change of phenylalanine to cysteine. As shown in Fig. 4, both of
GST-Glu
/Ser
Arg/Phe
His/Leu
Met and
GST-Glu
/Phe
Cys containing point
mutations in CSKH region had significant decreases in binding to Lck.
Finally, GST-Glu
/Arg
, which contained only
37 amino acids spanning CSKH and SH3B elements, was capable of binding
efficiently to Lck; GST-Glu
/Arg
exhibited
only a slight reduction of Lck-binding activity when compared with
GST-Glu
(Fig. 4).
Figure 3: CSKH is the additional motif required for Lck binding to Tip. Experimental procedures and exposure time were the same as described in Fig. 2. Box ``mSH3B'' represents the mutations of proline residues to alanines in the SH3B motif as described in Fig. 1. Boldface letters represent the amino acid sequences in the CSKH motif homologous to carboxyl termini of Src-related kinases. ++++, strong binding; -, no binding.
Figure 5:
CSKH, spacer, and SH3B motifs are
necessary and sufficient for efficient binding to Lck. Experimental
procedures were the same as described in Fig. 2. GST, Tip,
Tyr (Y
), Glu
(E
), Glu
/Arg
(E
/R
), and SH3B are
described in the text and in Fig. 2and Fig. 4. CSKH/SH3B
construct derived from GST-Glu
/Arg
contains
the deletion of 18 amino acids between CSKH and SH3B motif.
Glu
/Gly
construct contains the CSKH motif
and 18 intervening amino acid sequence without SH3B motif. The gel was
overexposed to show the lack of Lck-binding of CSKH/SH3B,
Glu
/Gly
, and SH3B fusion
proteins.
Figure 4:
Mutational analysis of CSKH motif.
Experimental procedures were the same as described in Fig. 2.
Glu/Ser
Arg/Phe
His/Leu
Met (E
/S
R/F
H/L
M)
and Glu
/Phe
Cys (E
/F
C)
contain the mutations as described in the context.
Glu
/Arg
(E
/R
) contains 37 amino
acids spanning CSKH and SH3B elements. Box SH3B represents the SH3B
element. ++++, strong binding; +, weak
binding.
18 amino acids that are
present between the CSKH and SH3B elements in Tip may link these two
elements. We thus analyzed the properties of a mutant with these
sequences deleted from the GST-Glu/Arg
construct. Overlapping oligonucleotides capable of encoding CSKH
and SH3B were fused to generate mutant construct, GST-CSKH/SH3B, which
is missing the coding sequences for the intervening amino acid residues
156-173. Again, GST-CSKH/SH3B fusion protein was mixed with
S-labeled cell lysates containing Lck protein.
GST-CSKH/SH3B mutant protein with the 18 amino acids deleted showed
dramatically diminished binding to Lck, while GST-Glu
and
GST-Glu
/Arg
bound efficiently to Lck under
the same conditions (Fig. 5). This demonstrates that the region
between CSKH and SH3B is required for efficient Lck binding and is
likely to function as a spacer between CSKH and SH3B elements.
Finally, we examined whether the CSKH motif and the intervening 18
amino acid region were capable of binding to Lck in vitro. To
study this, GST-Glu/Gly
fusion construct
containing CSKH and the 18-amino-acid intervening sequence without the
SH3B motif was generated, and binding activity to Lck in vitro was analyzed. As shown in lane6 of Fig. 5, GST-Glu
/Gly
was grossly
deficient for binding to Lck.
Thus, the CSKH, spacer and SH3B motifs are necessary and sufficient for Lck-binding in vitro.
Figure 6:
Association of Tip with Lck in insect
cells. Sf9 insect cells were infected with recombinant baculoviruses
expressing Tip and Lck as indicated at the bottom of the
figure. A, complex formation between Tip and Lck. After 48 h
of infection, cells were labeled with
[S]methionine. Cell lysates were used for
precipitations with anti-AU-1 (lanes1 and 2) and anti-Lck (lanes3 and 4)
antibody and immune complexes were separated by SDS-PAGE. Overnight
exposure. B, in vitro kinase assays of immune complexes of
anti-AU-1 and anti-Lck antibodies. Immunoprecipitations were performed
with anti-AU-1 (lanes1 and 2) and anti-Lck
antibody (lanes3 and 4). These immune
complexes were assayed for kinase activity with
[
-
P]ATP, and labeled proteins were
separated by SDS-PAGE, 2-s exposure. C, complex formation of
wild-type and mutant forms of Tip with Lck in insect cells. Mutant
Tip/Tyr
Ser (Y
S) and
Tip/mSH3B were described in the text. After 48 h of infection, cells
were labeled with [
S]methionine. Cell lysates
were used for precipitations with anti-AU-1 antibody. After
immunoprecipitation, proteins were separated by SDS-PAGE. The molecular
markers are ovalbumin (45 kDa) and bovine serum albumin (69
kDa).
Immune
complexes from insect cells were subjected to in vitro kinase
reaction. Anti-Lck immune complexes from Sf9 cells infected with Lck
baculovirus alone or coinfected with Lck and Tip baculoviruses were
used for the assay of in vitro kinase activity. 56-kDa
phosphorylated Lck was detected in both cells, while 42-43-kDa
phosphorylated Tip was additionally detected from Sf9 cells coinfected
with Lck and Tip baculoviruses (Fig. 6B, lanes3 and 4). The strongly phosphorylated
42-43-kDa protein in coinfected cells was shown to be Tip on the
basis of its presence only when Tip-expressing virus was included and
by its precipitation with the AU-1-tag antibody (Fig. 6B). AU-1 immune complexes from Sf9 cells
infected with Tip baculovirus alone showed weak phosphorylation of Tip
protein (Fig. 6B, lane1).
Phosphoamino acid assay of the weakly phosphorylated Tip in the absence
of Lck revealed phosphorylation mainly at serine and threonine
residues, suggesting the possible presence of serine and threonine
kinases in the AU-1 complexes (Fig. 7A). Lck kinase
activity in AU-1 immune complexes strongly phosphorylated both Lck and
Tip (Fig. 6B, lane2). Phosphoamino
acid assays of Tip phosphorylation in the presence of Lck showed that
Tip contained P-labeled phosphorylation predominantly at
tyrosine residues and a minor amount of phosphorylation at serine and
threonine (Fig. 7B). Lck demonstrated phosphorylation
only at tyrosine residues (Fig. 7C). The strong
phosphorylation of Tip and Lck shown in Fig. 6B was
detected with an exposure time of only 2 s. Migration of Tip protein by
SDS-PAGE was slightly retarded after association with Lck protein (Fig. 6, A and B, lane2).
This slower migration was likely due to the phosphorylation of Tip by
Lck.
Figure 7:
Two-dimensional phosphoamino acid analysis
of in vitro phosphorylated Tip and Lck proteins. Sf9 insect
cells were infected with recombinant Tip and/or recombinant Lck
baculoviruses. After 48 h of infection, cell lysates were used for
precipitations with anti-AU-1 (A and B) or anti-Lck (C) antibody. These immune complexes were assayed for kinase
activity with [-
P]ATP, and phosphorylated
proteins were subjected to phosphoamino acid analysis. Phosphoamino
acid analyses were performed with labeled Tip from insect cells
infected with Tip recombinant baculovirus (A), labeled Tip
from insect cells infected with Tip and Lck recombinant baculoviruses (B), and labeled Lck from insect cells infected with Tip and
Lck recombinant baculoviruses (C). S, phosphoserine; T, phosphothreonine; Y,
phosphotyrosine.
Since the SH3B motif of Tip is important for in vitro binding to Lck, two mutants of tip were generated and
expressed in Sf9 insect cells using baculovirus. The Tip/Tyr
Ser mutant contains the change of tyrosine at amino acid
number 114 to serine, and Tip/mSH3B has the changes of proline residues
within the SH3B motif to alanines as described in Fig. 1.
Wild-type Tip, Tip/Tyr
Ser, and Tip/mSH3B were
expressed in insect cells with or without Lck using the baculovirus
expression system. Tip/Tyr
Ser migrated slightly
faster than wild-type Tip in SDS-PAGE (Fig. 6C, lanes2 and 5). This was also detected in other cell
types including COS-1 and Rat-1 cells (data not shown). Coinfection of
Sf9 cells with Lck and wild-type or mutant Tip baculoviruses showed
that wild-type Tip and Tip/Tyr
Ser efficiently
formed complexes with Lck, while the Tip/mSH3B mutant showed a dramatic
decrease of binding activity with Lck (Fig. 6C).
After transfection of wild-type or mutant forms of tip gene
with lck, cells were labeled with
[S]methionine and
[
S]cysteine. Half of the cell lysates were used
for in vitro kinase assay after precipitation with anti-Lck
antibody, and the other half of the cells were used for
immunoprecipitation with anti-AU-1 antibody to show the level of Tip
expression. Immunocomplexes precipitated by anti-Lck antibody were
employed for in vitro kinase assay with
[
-
P]ATP to show association with and
phosphorylation of Tip. Mutations in SH3B and/or CSKH motifs greatly
diminished complex formation with Lck, while wild-type Tip was
efficiently associated with Lck in COS-1 cells (Fig. 8A). Under these conditions, similar amounts of
wild-type and mutant Tip were expressed in COS-1 cells (bottom of Fig. 8A). Thus, the SH3B and CSKH motifs are
important for association of Tip with Lck in COS-1 cells.
Figure 8:
In vivo association of Tip with
Lck in COS-1 and Rat-1 cells. A, association of Tip with Lck
in COS-1 cells. Expressed wild-type (wt) and mutant forms of
Tip were indicated at the bottom of the figure. COS-1 cells
were transfected with pFJ-Lck alone (lane2) or
together with pBKCMV expressing wild-type or various mutant forms of
Tip as indicated at the bottom of figure. After 48 h of
transfection, cells were labeled with
[S]methionine and
[
S]cysteine. Half of the cell lysates was used
for in vitro kinase assays of Lck immune complexes (top), and the other half of the cells was used for
immunoprecipitation with anti-AU-1 antibody to show the level of the
expression (bottom). Untransfected COS-1 cells (lane1) were used as a control. Labeled proteins were
fractionated by SDS-PAGE and detected by autoradiography. B,
complex formation between Tip and Lck in Rat-1 cells. Stably
transfected Rat-babe-Lck cells expressing wild-type Tip, Tip/mSH3B, or
Tip/
CSKH were established by transfection with pBKCMV constructs.
Expression of wild-type and mutant forms of Tip are indicated at the bottom of the figure. Rat-babe (lane1) and
Rat-babe-Lck (lane2) were used for controls. Lysates
of 1
10
cells were used for immunoprecipitation
with anti-Lck and anti-AU-1 antibodies. Immune complexes were subjected
to in vitro kinase assays with
[
-
P]ATP. Labeled proteins were fractionated
by SDS-PAGE and detected by autoradiography. The level of expression of
Tip was detected by immunoblot with whole cell lysates corresponding to
1
10
cells. Immunoblot detection was performed with
a 1:1000 dilution of primary AU-1 antibody and developed with ECL (bottom). Arrows indicate Lck and Tip
proteins.
tip and lck genes were also stably expressed in Rat-1
fibroblast cells. After transfection of Rat-1 cells with recombinant
retroviral vector pBabe-Lck, Rat-babe-Lck cell line was selected by
growth in medium containing 5 µg/ml of puromycin. Expression of Lck
in Rat-babe-Lck cells was confirmed by immunoblot with anti-Lck
antibody (data not shown). To express the wild-type Tip and mutant
forms of Tip, Rat-babe-Lck cells were transfected with pBKCMV
constructs containing wild-type Tip, Tip/mSH3B, or Tip/CSKH and
then selected with 500 µg/ml of G418. Similar amounts of tip gene expression were detected in these cells by immunoblot with
AU-1 antibody (bottom of Fig. 8B). To
investigate stable complex formation between Tip and Lck in Rat-1
cells, anti-Lck, and anti-AU-1, immune complexes were subjected to in vitro kinase assays. As shown in Fig. 8B,
mutant Tip/mSH3B and Tip/
CSKH showed a dramatic reduction in
complex formation with Lck in Rat-1 cells when compared with wild-type
Tip, which was efficiently associated with Lck under the same
conditions. In addition to Lck protein, 62- and 110-kDa phosphorylated
proteins were detected in anti-AU-1 immune complexes from Rat-1 cells
expressing wild-type tip gene (Fig. 8B). Thus,
SH3B and CSKH motifs are essential for in vivo complex
formation of Tip with Lck in COS-1 and Rat-1 cells consistent with the in vitro binding assays.
Tip protein encoded by the oncogenic herpesvirus saimiri strain C488 was previously shown to be expressed in virus-transformed T cells and to be associated with the major T cell tyrosine kinase Lck(21) . We have now identified sequences within Tip that are responsible for the efficient binding to Lck in vitro as well as in vivo. A segment of only 37 amino acids containing a region homologous to the carboxyl-terminal regulatory domain of Src-related kinases and a proline-rich putative SH3 binding site linked by a short spacer region were sufficient for efficient binding to Lck.
SH3 domains are small units of 55-70 amino acids found in
nonreceptor tyrosine kinases and other signaling molecules such as
phospholipase C, PI 3-kinase, and Grb2. They mediate
protein-protein interactions and also link these proteins to the
cytoskeletal architecture(3, 31) . The identification
of several SH3 binding proteins by expression cloning and affinity
chromatography has revealed that SH3 domains bind to short proline-rich
peptide motifs of 9 or 10 amino
acids(26, 27, 28) . Binding assays with
biased recombinatorial peptide libraries confirmed these findings and
defined the roles of the individual amino acid residues(26) .
The proline-rich region of Tip has high identity with an SH3 binding
consensus sequence and has been shown here to be essential for Lck
binding in vitro and in vivo. Thus, the presence of a
proline-rich region within Tip with high homology to known SH3 binding
sequences and its importance for the binding to Lck suggest that Tip is
likely to associate with Lck at least in part through its SH3 domain.
The 10-amino-acid CSKH motif in Tip has high homology with the
carboxyl terminus of Src-related kinases, which represents a part of
the conserved kinase domain XI and the regulatory region(32) .
It has 70-80% identity with the corresponding regions of Src,
Yes, Fyn, and Fgr; it has lower homology with that of Lck. Computer
searches with the CSKH sequence of Tip revealed a number of other
proteins with a high degree of homology; a mitochondrial F1 ATP
synthase chain and several genes encoding enzymes in carbohydrate
metabolism(33, 34, 35) . However, the role of
the homologous sequences in these proteins has not been studied.
Evidence is accumulating that the tyrosine protein kinase activity
of the Src family is regulated primarily through phosphorylation of the
carboxyl-terminal tyrosine residue such as Tyr for Src or
Tyr
for Lck(7, 25) . In addition,
several reports have suggested that the region surrounding the
carboxyl-terminal tyrosine residue is important for kinase activity and
protein interaction. For example, the addition or deletion of amino
acids from this region of the Src activates the kinase activity and
thus the transforming potential(36) , and this same region of
Src appears to be required for stable association with polyoma virus
middle T antigen (25) . These results suggest that the
carboxyl-terminal region may be involved in regulation of kinase
activity in the Src family by assisting with the interaction of the
phosphorylated carboxyl-terminal tyrosine to the binding pocket within
the SH2 domain. The presence of a segment within Tip with high homology
to the carboxyl terminus of Src-related kinases and its importance for
Lck binding suggest that the CSKH domain of Tip participates in the
interaction with Lck analogous to that described above, thereby
cooperating with the SH3B motif to enhance binding affinity to Lck.
Furthermore, it provides indirect experimental support for the
potential role of carboxyl-terminal sequences of individual Src kinases
in intramolecular interactions.
The 18-amino-acid segment linking the CSKH and SH3B domains does not share significant homology with any protein in the data base. However, it clearly influences binding of Tip to Lck as shown in Fig. 5. This stretch could possibly associate directly with Lck, or it may simply represent a linker sequence facilitating the alignment of CSKH and SH3B to their target sequences.
Quite a number of cellular proteins have been found to associate
with Lck. These include cell surface receptors like CD2, CD4, CD5, CD8
and interleukin 2
receptor(6, 37, 38, 39, 40) ,
downstream effectors like GPI anchored
proteins(41, 42, 43) , PI 3- and PI
4-kinases(44) , p95(45, 46) , Ras GAP(47) , and protein
kinases like Raf-related protein, (48) ,
ZAP-70(49, 50) , and Syk(51) . Tip could alter
the interaction of Lck with cellular substrates that physiologically
bind to the SH3 and/or SH2 domains of Lck. Alterations in complex
formation between cellular proteins and Lck may ultimately deregulate
signal transduction through Lck in transformed T cells expressing Tip.
Any role for Tip in altering T cell signal transduction in the
process of viral-induced cell growth transformation is yet to be
defined. The association of Tip with Lck could conceivably activate Lck
activity to achieve virus-induced T cell transformation similar to the
constitutive activation of Src by polyomavirus middle T
antigen(52) . Alternatively, the association of Tip may
interfere with normal Lck function by preventing its interaction with
substrates that normally bind to Lck. Analogous to Tip, LMP2A is
expressed in latently infected B lymphocytes by Epstein-Barr virus,
another member of the group of herpesviruses, and it associates
with the B cell tyrosine kinase Syk and Lyn(53) . LMP2A is not
necessary for B cell transformation by Epstein-Barr virus, but it has
been shown to block the effects of sIg cross-linking on calcium
mobilization, tyrosine phosphorylation, and reactivation of
Epstein-Barr virus from latent infection in the transformed human B
lymphocytes(53, 54) . If Tip were to function
analogously in T cells, we may expect it to block Lck-mediated signal
transduction. There is good evidence to indicate that an HSV-encoded
transforming protein, STP, acts downstream of Lck by binding to Ras and
activating the Ras pathway. (
)Direct activation of the Ras
pathway by STP may make Lck activation not only unnecessary for growth
transformation but also detrimental to the virus.