(Received for publication, September 1, 1994; and in revised form, November 15, 1994)
From the
The cytoplasmic protein tyrosine kinase p56 has been implicated as an effector of interleukin-2-induced
cell division in T-lymphocytes, but little is known about physiological
substrates for p56
during these events. We have
used p56
fusion proteins to identify potential
cytoplasmic signal transduction proteins that bind to p56
in mitotically activated human peripheral blood lymphocytes
and in constitutively dividing leukemic T-cell lines. In peripheral
blood lymphocytes, we have observed an interleukin-2-dependent tyrosine
phosphorylation of a 70-kDa protein and binding of tyrosine
phosphorylated p70 to the SH2 domain of p56
. A
70-kDa phosphoprotein was also observed to constitutively bind
p56
in leukemic T-cells. Affinity purification
of p56
-associated p70 and sequencing of
proteolytic fragments revealed identity to a 62-kDa protein that has
been identified as a ras-GTPase activating protein. These
results demonstrate a stimulation-dependent tyrosine phosphorylation of
p70 and its interaction with p56
and may provide
a link between p56
and GTPase-mediated signal
transduction pathways in activated T-lymphocytes.
Human peripheral T lymphocytes spontaneously arrest in a
quiescent (G) state during the process of maturation and
can be induced to re-enter the cell cycle in response to mitogenic
lectins or the T-cell growth factor interleukin-2 (IL-2) (
)(1, 2, 3, 4) . As the
IL-2 receptor does not possess intrinsic catalytic activity, the early
responses to IL-2 stimulation must be transmitted by
receptor-associated cytoplasmic enzymes. One possible candidate for an
IL-2 receptor-associated catalytic component is the T-cell-specific
tyrosine kinase p56
. Stimulation of T-cells with
IL-2 results in serine/threonine phosphorylation of p56
and induces a transient increase in p56
kinase activity(5) . In addition, there is a rapid
tyrosine phosphorylation of the IL-2 receptor
subunit following
IL-2 stimulation(6) . More direct evidence of p56
involvement in IL-2-mediated signal transduction comes from
coimmunoprecipitation experiments demonstrating an in vivo physical association between the IL-2 receptor
subunit and
p56
(7) . However, additional components
and downstream effectors of this signaling process remain to be
established.
The association of substrates or other signal
transduction components with many cytoplasmic protein tyrosine kinases
is often mediated by src homology (SH) domains found within
the amino-terminal half of all known src-like tyrosine
kinases(8) . The importance of these motifs in signal
transduction networks is also derived from the observation that SH2 and
SH3 domains are necessary components of many additional cellular
signaling molecules that are not members of the src family of
tyrosine kinases, such as the ras-GTPase activating protein
(GAP), the 85-kDa subunit of phosphatidylinositol-3-kinase and
phospholipase C(9, 10, 11) . Another
class of SH2 and SH3 containing proteins includes SEM-5, Drk, GRB-2,
Nck, and CRK, which have been termed adaptor proteins because they lack
catalytic activity and appear to link receptor tyrosine kinases to ras signaling(9, 10, 11, 12, 13, 14) .
We have used bacterially expressed p56 to
identify proteins that bind to this protein tyrosine kinase in human
T-cells activated by IL-2 or phytohemagglutinin (PHA). Our results
demonstrate an IL-2 or PHA stimulation-dependent tyrosine
phosphorylation of a 68-70-kDa protein in peripheral blood
lymphocytes (PBLs) and binding of the tyrosine phosphorylated form of
this protein to the SH2 domain of p56
.
Purification and sequence analysis of p70 showed that it was related to
the previously described p62, a ras-GAP and nucleic
acid-binding protein(15) . These results suggest that tyrosine
phosphorylation and interaction of p70 with p56
is an important early event in IL-2-induced onset of cell
cycle progression in T-lymphocytes.
Purification of p70 for microsequencing was carried out essentially
as for other in vitro association experiments with the
following modifications: 2 10
Molt-4 cells were
lysed in 10 ml of lysis buffer. The Molt-4 lysate was then mixed for 1
h at 4 °C with 30 µg of glutathione-agarose-bound pG-c221.
Following adsorption, the glutathione-agarose pellet was extensively
washed, resolved by SDS-PAGE, transferred to nitrocellulose membrane
(Schleicher and Schull), stained with ponceau-S (Sigma), and the 70 kDa
band excised. Tryptic digestion, HPLC separation and microsequencing of
the 70-kDa sample was done by the Harvard Microchemistry Facility
(Boston, MA).
Figure 1:
A (upper panel): depiction of the
p56-fusion protein constructs. The GST portion of the
fusion protein (not shown) is 27 kDa and is continuous with the amino
terminus of p56
. Numbers above the vertical arrows indicate amino acid residues. Lower
panel, the fusion proteins were purified by binding to
glutathione-agarose as described (17) and were resolved on a
10% SDS-polyacrylamide gel. Lane 1, GST; lane 2,
pG-wt; lane 3, pG-c323; lane 4, pG-st347; lane
5, pG-c275; lane 6, pG-c221; lane 7, pG-c117; lane X, Molecular mass markers of 97, 66, 45, and 31 kDa. B, a 70-kDa phosphotyrosine-containing protein associates with
p56
fusion proteins in lysates of activated
normal human peripheral blood lymphocytes. ST, untreated
cells; PHA, stimulation with PHA for 5 min; IL-2,
stimulation with IL-2 for 5 min. C, A 70-kDa
tyrosine-phosphorylated protein associates with p56 fusion proteins and
coimmunoprecipitates with p56. Lysates of Molt-4 cells containing 150
µg of total protein were incubated for 1 h at 4 °C with either
GST bound to glutathione-agarose or p56 fusion protein, pG-c221, bound
to glutathione-agarose. Fusion protein-bound and -unbound fractions
were isolated, resolved by SDS-PAGE, and immunoblotted with
anti-phosphotyrosine antibodies. Lane 1, whole cell lysate; lane 2, GST-unbound fraction; lane 3, pG-c211-unbound
fraction; lane 4, GST-bound fraction; lane 5,
pG-c221-bound fraction; lane 6, anti-p56 immunoprecipitate. D, the SH2 domain of p56 is required for association with
tyrosine-phosphorylated p70. COOH-terminal-deleted p56 fusion proteins
bound to glutathione-agarose were incubated with Molt-4 cell lysates
and the bound fraction isolated and immunoblotted with
anti-phosphotyrosine antibodies as above: lane 2, pG-c323; lane 3, pG-c275; lane 4, pG-c221; lane 5,
pG-c117. Lane 1 is 75 µg of Molt-4 whole cell lysate that
was not preincubated with fusion protein.
An additional phosphotyrosine-containing protein
of approximately 85 kDa was observed to associate with p56 in some experiments (Fig. 1C, lane 5).
We have previously demonstrated that phosphatidylinositol-3-kinase
activity from leukemic T-cell lines binds to the SH3 domain of
p56
(19) . Therefore, it is possible that the
broad anti-P-Tyr immunoreactive band at approximately 85 kDa (Fig. 1C, lane 5) represents the p85 subunit
of phosphatidylinositol-3-kinase. We are currently investigating this
possibility.
To assess the
phosphotyrosine dependence of the association between p56 and p70, we performed binding experiments using cell lysates
prepared in the presence (Fig. 2, A and B, lanes 1) or absence (Fig. 2, A and B, lanes 2) of phosphatase inhibitors. In the absence of
phosphatase inhibitors, the p56
-bound fraction from
T-cell lysates contained no detectable anti-P-Tyr immunoreactive
proteins (Fig. 2A, lane 2). However, when lck-associated proteins were detected by blotting with
biotinylated p56
, a reduced level of dephosphorylated p70
could still be observed in the lck-bound fraction (Fig. 2B, lane 2). Identical results were
obtained using cell lysates pretreated with phosphatase (Fig. 2C). These results indicate that p70 is able to
interact with p56
in a phosphotyrosine-independent manner
and suggest that a fraction of p70 is cabable of binding to either the
unique region or the SH3 domain of p56
. The
p56
-associated protein (Fig. 2, panel B)
with an apparent molecular mass of 65 kDa has not been identified, but
proteolytic digestion and comparison of fragment sizes with p70
indicate that p65 is not a dephosphorylated form of p70 (data not
shown). It is possible that p56
-associated p65
corresponds to the 65-kDa heterogenous nuclear ribonucleoprotein K
recently identified in src-p68 protein complexes(20) .
Figure 2:
p70 does not require phosphotyrosine for
association with lck fusion proteins. Molt-4 cells were lysed
in the presence (lane 1 of panels A and B)
or absence (lane 2 of panels A and B) of
phosphatase inhibitors NaVO
(1 mM) and p-nitrophenylphosphate (3.75 mg/ml). Lysates were then
incubated with p56
fusion protein pG-c221.
Fusion protein-bound fractions were isolated and protein complexes were
resolved by SDS-PAGE, transferred to nylon membrane, and immunoblotted
with anti-P-Tyr antibodies (panel A). Following anti-P-Tyr
immunoblotting, dephosphorylated p70 was detected by incubating filters
in the presence of biotinylated p56
as described
under ``Materials and Methods'' (panel B).
Biotinylated p56
was also used to detect
p56
-bound proteins from T-cell lysates that had
been pretreated with phosphatase (lane 2, panel C) or
untreated controls (lane 1, panel
C).
Figure 3:
Sequencing of proteolytic fragments from
purified p70 reveal sequence identity with GAP-associated p62 (15) . Amino acid sequence from two HPLC fractions of tryptic
peptides generated from p70 isolated by affinity chromatography using
p56 fusion protein. Alignment of p70 peptide
sequences with GAP-associated p62 sequence as given in (15) .
To further characterize the association
of p70 with endogenous p56, we analyzed anti-p56
immunoprecipitates and p56
fusion
protein-associated molecules by immunoblotting with anti-p62 sera.
Anti-p56
immunoprecipitates contained a protein at 70 kDa
that cross-reacted with anti-p62 sera (Fig. 4A, lane 1). The 70-kDa phosphotyrosine-containing protein from
T-cell lysates that bound to bacterially expressed p56
was also recognized by the anti-p62 sera (Fig. 4A, lane 3). Whole cell lysates from
human leukemic T-cells contained an anti-p62 immunoreactive band (Fig. 4B, lane 2) at approximately 70 kDa that
comigrated with the p62 immunoreactive protein associated with
bacterially expressed p56
(Fig. 4B, lane 1). A p62 immunoreactive band was not observed in control
immunoprecipitates utilizing non-immune rabbit sera (Fig. 4A, lane 2). Identical results were
obtained with IL-2-activated PBLs (data not shown).
Figure 4:
A) p56-associated
p70 cross reacts with anti-p62 sera. Lysates of the human leukemic
T-cell line Molt-4 (400 µg) were incubated with
anti-p56
sera cross-linked to protein
A-Sepharose (lane 1) or non-immune sera cross-linked to
protein A-Sepharose (lane 2) or p56
fusion protein (pG-c275) bound to glutathione-agarose (lane 3). The bound fraction was isolated by centrifugation,
washed, and resolved by SDS-PAGE. Proteins were transferred to
polyvinylidene difluoride membrane and immunoblotted with anti-p62 sera
(Santa Cruz Biotech.) Immunoreactive bands were detected using enhanced
chemoluminescence (Amersham). B, anti-p62 immunoblot. Lane
1, molt-4 whole cell lysate (50 µg). Lane 2,
p56
fusion protein (pG-c275)-bound fraction
following incubation with 400 µg of Molt-4
lysate.
Figure 5:
p70
does not coimmunoprecipitate with ras-GAP. A,
anti-p62 immunoblot of whole cell lysates (50 µg) from human
fibroblasts (lane 1), NIH 3T3 mouse fibroblasts (lane
2), SRA-transformed CEFs (lane 3), Molt-4 cells (lane
8), or anti-ras-GAP immunoprecipitate of SRA/CEFs (lane 4), anti-p62 immunoprecipitate from SRA/CEFs (lane
5), the p56-bound fraction (pG-c221) from
SRA/CEFs (lane 6), and the p56
-bound
fraction (pG-c221) from Molt-4 cells. B, anti-p62 immunoblot
of Molt-4 whole cell lysate (25 µg) (lane 2), the
p56
-bound (pG-c275) fraction following
incubation with 300 µg Molt-4 lysate (lane 3), preimmune
sera immunoprecipitate (lane 1) or anti-GAP immunoprecipitate
from 300 µg of Molt-4 lysate (lane 4), or 2 µg of
bovine serum albumin (lane 5). NIS, non-immune sera; WCL, whole cell lysate; F-P, fusion protein-bound
fraction.
Human peripheral blood T-lymphocytes are a good source of
naturally synchronized cells that have been used to describe the
ordered activation of cyclin-dependent kinases during the G1 to S
transition of the cell cycle(3, 4, 21) . Most
recently, IL-2 has been shown to down regulate p27kip1, an inhibitor of
cyclin-dependent kinase 2(2) . We have found that an early
event in IL-2- or PHA-induced cell cycle progression in T-cells
involves phosphorylation of p70 and association of phosphorylated p70
with the SH2 domain of p56. Recently, a similar molecule
has been shown to associate with activated c-Src in mitotic
fibroblasts but not in asynchronously growing
cells(20, 22) , indicating that p70 phosphorylation
and interaction with src family tyrosine kinases may be a
common regulatory feature of cell cycle progression. Amino acid
sequence data indicated that p56
-associated p70 is either
equivalent or closely related to the GAP-associated p62. However, we
have not observed p70 in association with anti-GAP immunoprecipitates
from T-cell lysates. In addition, we have not observed anti-GAP
immunoreactive proteins in p56
immunoprecipitates or
associated with p56
fusion proteins following incubation
with T-cell lysates (data not shown). These observations are consistent
with the recent observations that src-associated p68 also does
not bind to GAP(20, 22) . Consequently, it is likely
that p70 represents the product of an alternatively spliced or
post-translationaly modified form of p62 that does not associate with ras-GAP.
We have observed that the tyrosine phosphorylated
form of p70 binds to the SH2 domain of p56. However, we
have also observed that a proportion of dephosphorylated p70 remains
bound to p56
and probably interacts with p56
in an SH2-independent fashion. Although we have not demonstrated
binding of dephosphorylated p70 to the SH3 domain of p56
,
we feel that SH3-directed association of dephosphorylated p70 is likely
because GAP-associated p62 (15) contains proline-rich sequences
that may mediate SH3 binding(23) , and because p68 has been
shown to interact with the SH3 domains of both src and fyn(20, 22) . Our observation of
SH2-dependent, as well as SH2-independent association between
p56
and p70, together with the recent demonstration of
SH3-directed association of p68 to src and fyn(20, 22) suggests that tyrosine kinase-p70
complexes may exist in distinct pools within the cell. It will be
interesting to determine if there are separate pools of
p56
-p70 complexes and, if so, to ascertain whether or not
the relative size of each fraction is determined by the activation
state of p56
.
The identity of the 110-kDa
phosphoprotein binding to p56 in IL-2- and PHA-stimulated
PBLs has not been investigated. However, it is possible that this
protein is the microtubule-associated GTPase, dynamin, that is known to
bind SH3 domains (24) and has also been observed to coassociate
with src-p68 complexes(20) . It remains to be
determined as to why p110 was observed to associated with p56
in lysates of activated PBLs but not in lysates of leukemic
T-cells. It is possible that p110 from leukemic T-cells either did not
bind to p56
or that binding was not detected because p110
was not tyrosine-phosphorylated. In either case this could be an
important distinction between normal versus leukemic T-cells.
The physiological significance of p70 binding to p56 in activated lymphocytes is not known. Although GAP-associated
p62 has homology to RNA-binding proteins and can bind RNA(15) ,
its function is not known. Additionally, it is possible that p70 does
not share a functional homology with GAP-associated p62, as p70 and src-associated p68 do not associate with GAP(20) .
However, evidence is accumulating which links signal transduction
through receptor and non-receptor tyrosine kinases to a family of small
GTPase proteins(25, 26, 27) . Although we did
not observe association between p70 and GAP, it is possible that p70
binds to additional GTPase-regulating proteins. Others have observed a
32-kDa protein with GTPase activity in association with CD4-p56
and CD8-p56
T-cell receptor complexes(28) .
Consequently, it is possible that the p56
-p70 complex may
interact with GAP-like proteins distinct from ras-GAP.
In
conclusion, several members of the src family of non-receptor
tyrosine kinases have been implicated in regulating aspects of T-cell
signaling by virtue of their ability to interact with the IL-2 receptor (7) or components of the T-cell antigen receptor
complex(29, 30, 31, 32) . Evidence
derived from other cell systems also indicates that src-like
tyrosine kinases may participate within multi-enzyme signal
transduction complexes in which many interactions are regulated by SH2
domains and tyrosine phosphorylation (8, 16) . Our
observation that a 70-kDa phosphotyrosine-containing protein bound to
the SH2 domain of p56 following IL-2 or PHA stimulation
of PBLs may indicate a GTPase-linked component of tyrosine
kinase-mediated signaling in mitotically activated T-cells. The
stimulation-dependent phosphorylation of p70 and association with
p56
in PBLs contrasts with the constitutive tyrosine
phosphorylation of p56
-associated p70 in human leukemic
T-cells and may indicate an important difference related to IL-2
independent growth of leukemic T-cell lines.