©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
p70 Phosphorylation and Binding to p56 Is an Early Event in Interleukin-2-induced Onset of Cell Cycle Progression in T-lymphocytes (*)

(Received for publication, September 1, 1994; and in revised form, November 15, 1994)

Lee B. Vogel(§)(¶) Donald J. Fujita (1)

From the From CNRS, Station Biologique, 29680-Roscoff, France and the University of Calgary Medical Centre, 3330 Hospital Drive N. W., Calgary, Alberta T2N-4N1, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Human peripheral T lymphocytes spontaneously arrest in a quiescent (G(0)) 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)(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 beta 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 beta 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.


MATERIALS AND METHODS

Fusion Protein Constructs

A human lck cDNA clone was used to prepare deleted constructs that were expressed from the bacterial expression vector PGEX-2T (Pharmacia). Each of the cDNA clones was isolated from Blue Script (Stratagene) as an NcoI-EcoRI fragment and ligated into SmaI-EcoRI-digested PGEX-2T or PGEX-3X. The construct designated pG-wt encodes the full-length wild type human lck. Constructs pG-c323, pG-c275, pG-c211, and pG-c117 were all deleted from the 5` end to codons 323, 275, 221, and 117, respectively, by digestion with exonuclease III. Each of these constructs was ligated into SmaI-EcoI-digested PGEX-2T. Construct pG-st347 was derived from an alternatively spliced lck cDNA clone encoding p56 SH2 and SH3 domains but not the kinase domain. (^2)All deletions and mutations were verified by nucleotide sequence analysis prior to PGEX subcloning. Expression of fusion protein was induced by the addition of 0.1 mM isopropyl-beta-D-thiogalactoside (Sigma) for 3 h. Harvesting and purification of the fusion proteins by affinity to glutathione agarose was carried out essentially as described by Smith and Johnson (17) . Appropriate expression of lck fusion proteins was verified by immunoblotting with anti-lck sera 1.7a. Sera 1.7a is an amino-terminal directed rabbit polyclonal antisera that was developed using the fusion protein pG-st347 as antigen.

Cell Culture

PBLs were isolated from ``buffy layer'' blood samples obtained from the Canadian Red Cross. Isolation of T-cells from these samples was accomplished in the following manner. White cells were separated from erythrocytes by centrifugation at 1500 times g for 10 min. The white cell layer was washed several times in 10 times volume of phosphate-buffered saline (PBS) to remove platelets. Finally, T-cell were separated from B-cells, monocytes, and macrophages on a percol gradient(18) . Following isolation, PBLs were cultured for 24 h in RPMI 1640, 10% fetal calf serum supplemented with 5 µg/ml PHA (Sigma). After induction with PHA, PBLs were expanded for 48-72 h in the presence of human recombinant IL-2 (Sigma) 20 units/ml. PBLs were grown in the absence of IL-2 for 48 h prior to induction experiments. The human cell line, Molt-4, was obtained from the American Type Culture Collection and was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Chicken embryo fibroblasts (CEF) were prepared from 11-day-old C/E chf- embryos (SPAFAS Inc., Norwich, CT) and maintained in Dulbecco's modified Eagle medium supplemented with 5% calf serum, 10% tryptose phosphate broth, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures of secondary CEFs were transformed by mass infection (multiplicity of infection > 1) with low passage stock of Rous sarcoma virus (strain, SRA). The infected cells were passaged three times within 10 days to ensure complete infection. SRA-transformed CEFs were maintained in the same medium as for CEFs with the addition of 0.6% Me(2)SO. For experiments, transformed cells or CEFs were seeded and grown to approximately 80% confluent then washed with PBS and lysed with Nonidet P-40 lysis buffer as described for T-cells.

Association with lck Fusion Proteins

Cells were lysed at 10^7/ml in ice-cold lysis buffer (0.15 M NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40, 2% glycerol, 1 mM Na(3)VO(4), 50 µg/ml leupeptin, 25 µg/ml aprotinin, 3.75 mg/ml p-nitrophenylphosphate) for 15 min on ice. Lysates were cleared by centrifugation at 13,000 times g for 5 min. In vitro association with immobilized p56 fusion protein was carried out as follows. Glutathione-agarose-bound fusion protein (50 pmol of fusion protein) was added to 150 µl (300 µg of total protein) aliquots of cell lysate and mixed at 4 °C for 1 h in a total volume of 300 µl. The glutathione-agarose-bound fusion protein was then collected by centrifugation and washed three times with cold PBS, 1% Nonidet P-40. The washed fusion protein pellets were then boiled in PAGE sample buffer and resolved by SDS-PAGE electrophoresis, transferred to nitrocellulose, and immunoblotted with affinity purified anti-phosphotyrosine antibodies. Immunoblotted filters were then incubated with I-labeled donkey anti-rabbit sera (Amersham) then autoradiographed at -80 °C. Other sera used for immunoblotting include anti-p62 sera (Santa Cruz Biotech, Santa Cruz, CA) and anti-ras-GAP sera (kindly provided by F. McCormick). Filters were also blotted with a biotinylated p56 fusion protein. Fusion protein construct pG-c221 was coupled at room temperature for 3 h with biotinidocaproate-N-hydroxysuddinimide ester (100 µg/ml, Sigma) at a fusion protein concentration of 2 mg/ml in 0.1 M sodium borate, pH 8.8. Biotinylated fusion protein was purified by extensive dialysis in PBS, 1 mM dithiothreitol and used at a concentration of 2 µg/ml in Tris-buffered saline, pH 8.0, 0.05% Tween-20. Biotinylated probes were detected with avidin-conjugated horseradish peroxidase at 1 µg/ml in Tris-buffered saline, pH 8.0, 0.05% Tween-20 and developed using enhanced chemoluminescence (Amersham). Phosphatase treatment of cell lysates was carried out at room temperature for 30 min using alkaline phosphatase (Boehringer) at 25 units/ml in lysis buffer without the addition of Na(3)VO(4) or p-nitrophenylphosphate.

Purification of p70 for microsequencing was carried out essentially as for other in vitro association experiments with the following modifications: 2 times 10^8 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).


RESULTS

T-cell Activation-induced Tyrosine Phosphorylation of p70 and Association with lck

To further examine early events during the re-entry of T-cells into the cell cycle, bacterially expressed p56 fusion proteins (Fig. 1) were employed to assay for p56-binding proteins in lysates of IL-2 or PHA-stimulated PBLs. Glutathione-agarose-bound p56 fusion protein or glutathione S-transferase (GST) alone were incubated with lysates of PBLs, and associated proteins were identified by anti-phosphotyrosine (anti-P-Tyr) immunoblotting (Fig. 1B). No p56-associated anti-P-Tyr immunoreactive proteins could be detected in lysates of PBLs that had been deprived of IL-2 for 48 h (Fig. 1B, lane 1 (ST)). However, when PBLs were stimulated with IL-2 or PHA for 5 min prior to lysis, a prominent p56-associated anti-P-Tyr immunoreactive protein of approximately 70 kDa was detected (Fig. 1B, lanes 2 (PHA) and 3 (IL-2)). Experiments using cells metabolically labeled with [P]orthophosphate demonstrated that lck-associated p70 became phosphorylated within 2 min following IL-2 stimulation (data not shown). Although less prominent, a 110-kDa p56-associated anti-P-Tyr immunoreactive protein was also observed in IL-2- or PHA-stimulated PBLs (Fig. 1B, lanes 2 (PHA) and 3 (IL-2)). Bacterially expressed GST did not bind to any anti-P-Tyr immunoreactive proteins in lysates from the human T-cell line Molt-4 (Fig. 1C, lane 4) or from lysates of PBLs (data not shown).


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.



p70 Is Constitutively Phosphorylated in Leukemic T-cell Lines

To further assess the growth factor dependence for tyrosine phosphorylation and p56 binding of p70, we performed p56-binding assays using lysates from an IL-2-independent human leukemic T-cell line, Molt-4. Lysates from Molt-4 cells contained a single prominent anti-P-Tyr immunoreactive protein migrating with an apparent molecular mass of 58 kDa (Fig. 1C, lane 1), and several fainter signals ranging from 70 to approximately 200 kDa (lane 1, Fig. 1, C and D). Molt-4 cell lysates were mixed with bacterially expressed p56 or control proteins immobilized on agarose, then separated into bound and unbound fractions. Following incubation with p56 fusion protein (pG-c211) the unbound fraction of Molt-4 lysate was depleted of an anti-P-Tyr immunoreactive protein at approximately 70 kDa (Fig. 1C, lane 3). A 70-kDa phosphotyrosine-containing protein was observed in the p56-bound fraction from Molt-4 lysates but was not observed in the GST-bound fraction (Fig. 1C, lanes 4and 5). In addition, immunoprecipitates of p56 contained an anti-P-Tyr immunoreactive protein at approximately 70 kDa (Fig. 1C, lane 6) indicating that the observed association between p56 fusion proteins and p70 likely reflects a physiological interaction. The observation that p56 association or tyrosine phosphorylation of p70 is not dependent on IL-2 or PHA stimulation in Molt-4 lysates, and the absence of a 110-kDa p56-associated phosphoprotein (Fig. 1C, lane 5) may represent important differences between leukemic and normal T-cells.

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.

p70 binds to the SH2 Domain of p56

To identify the p56 sequences required for association with the 70-kDa phosphoprotein, an overlapping series of carboxyl-terminal-deleted p56 fusion proteins were mixed with T-cell lysates, and p56-bound proteins were analyzed by immunoblotting with anti-P-tyr sera. The lck fusion proteins pG-c323 and pG-c275 both encode for the amino-terminal half of p56 including the unique region, the SH3 domain, the SH2 domain, and 122 or 54 amino acids, respectively, of the catalytic domain (Fig. 1A). Deleting either a portion or the entire catalytic domain from p56 had no effect on the ability of p70 to associate with p56 (Fig. 1D, lanes 2-4). However, when 104 amino acids comprising the p56-SH2 domain were deleted, p70 was not detected in association with p56 (Fig. 1D, lane 5). These results suggest that the tyrosine phosphorylated form of p70 requires the SH2 domain for association with p56.

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 Na(3)VO(4) (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).



p70 Is Closely Related to Gap-associated p62

Proteins binding to p56 were isolated from Molt-4 lysates by affinity purification with p56 fusion protein pG-c221. Two prominent protein bands of 65 and 70 kDa were observed by staining with Coomassie Blue. These two proteins were observed to bind specifically to p56 fusion proteins and not to GST alone or other GST constructs (data not shown). The 70-kDa p56-associated protein was isolated as described under ``Materials and Methods,'' and two tryptic peptides were sequenced to identify a total of 25 amino acids with a high degree of confidence. Both peptide sequences (Fig. 3) revealed complete identity with the GAP-associated p62 sequence derived from a human placental cDNA(15) .


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.



p70 Does Not Coimmunoprecipitate with ras-Gap

p62 was first identified as a GAP-associated protein in v-src-transformed fibroblast cell lines(15) . To investigate the apparent difference in molecular weight between GAP-associated p62 and lck-associated p70, we examined p62 immunoreactive proteins in human and mouse fibroblast cell lines and v-src-transformed CEFs. Additionally, to determine if lck-associated p70 could interact with GAP we examined anti-GAP immunoprecipitates from T-cell lysates for the presence of p62 immunoreactive proteins. Three major p62 immunoreactive proteins were observed in lysates from a human fibroblast cell line including a protein of approximately 70 kDa (Fig. 5A, lane 1). In lysates of mouse fibroblasts (NIH-3T3) only one p62 immunoreactive protein at approximately 70 kDa was observed (Fig. 5A, lane 2). Lysates from src-transformed CEFs also contained only one p62 immunoreactive protein, but this protein migrated with an apparent molecular mass of approximately 65-66 kDa (Fig. 5A, lane 3). The p62 immunoreactive protein in CEFs was immunoprecipitated with both anti-p62 serum as well as anti-GAP serum and also associated with p56 fusion protein (Fig. 5A, lanes 4-6). In human leukemic T-cell lysates (Molt-4) (Fig. 5A, lane 8), the most prominent anti-p62 immunoreactive protein was observed at approximately 70 kDa. This 70-kDa protein from Molt-4 whole cell lysates comigrated with the most prominent p62 immunoreactive protein associated with bacterially expressed p56 (Fig. 5A, lane 7). The p56-bound fraction also contained a p62 immunoreactive protein at approximately 65 kDa that comigrated with the p62 immunoreactive protein from anti-GAP and anti-p62 immunoprecipitates from SRA transformed CEFs. Upon longer exposure a p62 immunoreactive protein could also be observed at this position in whole cell lysates from Molt-4 cells (data not shown). However, anti-GAP immunoprecipitates from Molt-4 cells did not contain either a 65- or a 70-kDa p62 immunoreactive protein (Fig. 5B, lane 4). Consequently, the observation of a 65-kDa p62 immunoreactive protein in anti-GAP and anti-p62 immunoprecipitates, as well as in the p56-bound fraction from SRA-transformed CEFs and Molt-4 cells, suggests that there is more than one form of p62. Alternatively, these observations may indicate a nonspecific cross-reactivity of the p62 serum. Bovine serum albumin is an obvious contaminant of tissue culture samples that migrates on SDS-PAGE with an apparent molecular mass of 66 kDa. However, anti-p62 sera was not observed to cross-react with bovine serum albumin (Fig. 5B, lane 5).


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported during part of this work by a Medical Research Council Biotechnology Training Program studentship. To whom correspondence should be addressed. Tel.: 33-98-29-23-39; Fax: 33-98-29-23-42.

(^1)
The abbreviations used are: IL-2, interleukin-2; PBLs, peripheral blood lymphocytes; GAP, GTPase-activating protein; PHA, phytohemagglutinin; CEF, chicken embryo fibroblasts; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; GST, glutathione S-transferase; anti-P-tyr, anti-phosphotyrosine.

(^2)
L. B. Vogel, D. Fujita, and R. Arthur, manuscript submitted for publication.


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