1995 pp. 2313-2319
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CD45 Protein-tyrosine Phosphatase Is Specifically Associated with a 116-kDa Tyrosine-phosphorylated Glycoprotein (*)

(Received for publication, July 8, 1994; and in revised form, October 24, 1994)

Christopher W.Arendt (§)Hanne L.Ostergaard (¶)

From the Department of Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD45 is a protein-tyrosine phosphatase expressed on all cells of hematopoietic origin. In an attempt to further characterize CD45 function, we set out to identify molecule(s) that specifically associate with CD45. A 116-kDa protein was detected in immunoprecipitates from CD45 cells but not CD45 cells. The association between CD45 and this 116-kDa protein can be reconstituted by mixing lysates from CD45 cell lines with purified CD45. p116 appears to associate with CD45 through the external, transmembrane, or membrane-proximal region of CD45 since p116 is associated with a mutant form of CD45 possessing a truncated cytoplasmic domain. The association of p116 with CD45 is not isoform-specific as p116 associates equally well with various CD45 isoforms. We have determined that p116 is a tyrosine-phosphorylated glycoprotein and that it is associated with CD45 in all hematopoietic cells examined. Because of its broad distribution, it is possible that identification of p116 will provide additional insight into the function of CD45 in lymphoid as well as non-lymphoid hematopoietic cells.


INTRODUCTION

CD45 is a high molecular weight glycoprotein abundantly expressed on nucleated cells of hematopoietic origin(1) . Isoforms of CD45 arise from differential splicing of 4 exons (exons 4-7) encoding sequences found near the amino terminus of the molecule, giving rise to structurally related molecules differing in molecular weight. Different cell lineages express different isoforms, and the isoform usage often changes as cell differentiate. All isoforms of CD45 share a large, highly conserved cytoplasmic domain containing two subdomains. These subdomains have significant homology with human placental protein-tyrosine phosphatase 1B(2) , and it is well established that CD45 has intrinsic protein-tyrosine phosphatase activity(3, 4) .

CD45 has been shown to be important for both T- and B-lymphocyte activation. CD45-deficient T-cell lines are unable to be activated to proliferate, secrete interleukin-2, or mediate cytolytic events after stimulation through the T-cell antigen receptor(5, 6, 7) . CD45 appears to exert its function early in the activation cascade since tyrosine phosphorylation, generation of inositol phosphates, and an increase in intracellular Ca after stimulation through the T-cell or B-cell receptor for antigen are ablated in most CD45-deficient cells (8, 9, 10, 11) . Transfection of chimeric molecules containing the cytoplasmic domain of CD45 into CD45 cells restores the signaling capacity, suggesting that the external domain is not required for these early activation events(12, 13, 14) . CD45 appears to regulate the phosphorylation state and the enzymatic activity of src-related tyrosine kinases including p56 and p59(4, 15, 16, 17, 18, 19) , consistent with it being required early in the activation cascade. It is not clear if CD45 has additional substrates or if the regulation of src-related kinases is its only function during lymphocyte activation.

In an attempt to further define the role of CD45 in lymphocyte activation, a number of investigations have been undertaken to identify molecules that are specifically associated with CD45. Such associated molecules might regulate the enzymatic activity or modulate the physical proximity of CD45 with its specific substrate(s). The most commonly employed approaches include chemical cross-linking and cocapping. Using the same chemical cross-linking reagent, one group has demonstrated that CD45 immunoprecipitates contain CD2 (20) and another Thy-1 and the T-cell antigen receptor(21) . Cocapping studies suggest that the association of CD45 with CD4, CD8, LFA-1, and CD2 is isoform-specific(22, 23) . It has also been reported that CD45 is coimmunoprecipitated with CD26 (dipeptidyl peptidase IV) from digitonin lysates of human T-cells(24) . Since the expression of these various molecules is restricted to certain cell lineages, we speculate that there may be alternative molecules associated with CD45 in other cell types or that there may be more ubiquitously expressed molecules associated with CD45 in all hematopoietic cells.

Studies have also been performed to determine if there are protein interactions with the cytoplasmic domain of CD45 that may regulate its function. CD45 is associated with a 30-kDa protein (25) that has no homology with known sequences and appears to be expressed in all leukocytes(26) . This molecule has been postulated to be an adapter molecule for CD45-mediated signaling events. The cytoskeletal protein fodrin has also been shown to associate with CD45 and may stimulate its enzymatic activity(27) . The interaction of CD45 with the cytoskeleton, through fodrin, may also regulate its association with other cell-surface molecules(27) .

Our goal was to identify molecules that are stably associated with CD45 in the detergent Nonidet P-40. Herein we establish that a 116-kDa protein is associated with CD45 in immunoprecipitates of CD45 but not CD45 T-lymphomas. We also demonstrate that a 116-kDa protein appears to be specifically associated with CD45 in all CD45-expressing cells examined. Initial characterization of this molecule suggests that it is a tyrosine-phosphorylated glycoprotein. Identification of this 116-kDa protein might provide additional insight into the function of CD45 in non-lymphoid cells as well as lymphoid cells.


MATERIALS AND METHODS

Cell Lines and Antibodies

BW5147 and SAKRTLS 12.1 are mouse T-lymphomas. These cells and their CD45 variants as well as the BW5147 revertant have been described previously(4) . The 2 cells infected with pARV-1 retroviral constructs expressing isoforms of CD45 containing different sequences encoded by alternatively spliced exons have also been described(28) . These cells and all other cell lines were grown in Dulbecco's modified Eagle's medium with high glucose (Life Technologies, Inc.) containing 8% heat-inactivated defined calf serum (Hyclone, Logan, UT) and 2 mM glutamine.

The monoclonal antibodies used in this study were I3/2 (29) and M1/9.34 HL2, which are both specific for a portion of the ectodomain common to all isoforms of CD45, and M1/42, which is specific for mouse major histocompatibility complex class I molecules. Both M1/9.34 HL2 and M1/42 were obtained from ATCC. Cells producing monoclonal antibodies were grown in serum-free media (protein-free hybridoma medium II, Life Technologies, Inc.) and concentrated by ammonium sulfate precipitation followed by dialysis. The purity of the antibodies was verified by SDS-PAGE. (^1)The antibodies were then directly coupled to cyanogen-activated Sepharose 4B. The polyclonal antiserum to recombinantly expressed cytoplasmic domain of CD45 has been described(4) .

Metabolic Labeling and Surface Biotinylation

For metabolic labeling with [S]methionine, cells were incubated for 4 h at 10^7 cells/ml with 0.2 mCi/ml of TranS-label (ICN, Irvine, CA) in methionine-free RPMI 1640 medium containing 10% dialyzed calf serum. Cells were washed 3 times in cold Dulbecco's PBS (Life Technologies, Inc.) before lysis. Cells were prepared for surface biotinylation by washing twice in PBS and resuspending at 4 times 10^7 cells/ml in PBS containing 120 µg/ml Sulfo-NHS-Biotin (Pierce, Rockford, IL). Labeling was allowed to proceed for 10 min at room temperature and was quenched by incubation with an equal volume of culture medium for 5 min at 4 °C. Cells were then washed once in cold culture medium and 3 times in cold Dulbecco's PBS. Lysis and immunoprecipitation were performed as described below.

Preparation of Cell Lysates and Immunoprecipitates

Cells were washed 3 times in Dulbecco's PBS and lysed by resuspension in 0.5% Nonidet P-40, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM Tris-HCl, pH 7.6, unless otherwise noted, at 4 times 10^7 cells/ml. After 20 min on ice, postnuclear extracts were added to Sepharose 4B that had been previously coupled to antibody and blocked with 3% bovine serum albumin. After 45-90 min at 4 °C, immunoprecipitates were washed 3-6 times in lysis buffer. Reducing sample buffer was added directly to the beads and, after boiling, proteins were subjected to electrophoresis.

Polyacrylamide Gel Electrophoresis and Immunoblotting

Proteins were electrophoresed on either 7.5 or 10% gels by the Laemmli method(30) . Gels were either silver stained or proteins were transferred to Immobilon-P (Millipore Corp., Bedford, MA) in 20% methanol, 20 mM Tris base, 96 mM glycine for 2-3 h at 60 V. After transfer, proteins were stained with India ink as described in the Immobilon-P product bulletin. Biotinylated proteins were detected by incubation with Streptavidin-peroxidase (Boehringer Mannheim Canada, Laval, Quebec) at 1:30,000 followed by enhanced chemiluminescence (DuPont NEN) as described in the product manual. Tyrosine-phosphorylated proteins were revealed by enhanced chemiluminescence using affinity-purified rabbit anti-phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY) at 1.5 µg/ml followed by goat anti-rabbit coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Reconstitution of Association using Purified CD45

CD45 was purified by immunoaffinity chromatography by passing Nonidet P-40-solubilized SAKRTLS 12.1 extracts over an I3/2 column. After extensive washing in 0.5% Nonidet P-40, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.6, followed by 0.5% sodium deoxycholate, 0.15 M NaCl, 20 mM Tris-HCl, pH 7.6, which disrupts the association with p116, CD45 was eluted with 0.1% Nonidet P-40, 0.5 M NaCl, 50 mM sodium acetate, pH 4.0. One-ml fractions were immediately neutralized, and positive fractions were identified by enzyme-linked immunosorbent assay. Peak fractions were analyzed by SDS-PAGE revealing CD45 as the only species visible by silver staining.

For the reconstitution studies, membranes from CD45 SAKRTLS 12.1 cells were isolated as described previously(31) . Briefly, the cells were disrupted by nitrogen cavitation in PBS followed by high speed centrifugation to separate cytosolic proteins from the membrane fraction. Cell membranes were reconstituted at 1.6 mg/ml in lysis buffer, and an additional ultracentrifugation step was performed to eliminate the detergent-insoluble material. Samples were saved at each step for I3/2 immunoprecipitation in the presence or absence of approximately 10 µg of purified CD45. Beads mixed with detergent-solubilized fractions were prepared for SDS-PAGE by washing 5 times in lysis buffer containing 0.5 M NaCl and 3 times in regular lysis buffer. Beads treated with cytosolic proteins in the absence of detergent were washed 8 times in PBS.

Endoglycosidase F treatment

Immunoprecipitates were incubated overnight at 37 °C with 0.3 units of endoglycosidase F/N-glycosidase F (Boehringer Mannheim) in 50 µl of 0.25 M sodium acetate, pH 6.5, 20 mM EDTA, 1% 2-mercaptoethanol. The immunoprecipitates were washed 3 times in lysis buffer, resuspended in reducing sample buffer, and subjected to SDS-PAGE.


RESULTS

CD45 Specifically Associates with a Molecule of About 116 kDa in T-lymphoma Cell Lines

To determine if any proteins specifically immunoprecipitate with CD45, lysates were prepared from CD45 and CD45 SAKRTLS 12.1 T-lymphomas using buffer containing 0.5% Nonidet P-40. CD45 was immunoprecipitated with the CD45-specific monoclonal antibody I3/2 directly coupled to Sepharose 4B, washed extensively with lysis buffer, and subjected to SDS-PAGE. To minimize nonspecific interactions, the Sepharose 4B was blocked with 3% bovine serum albumin prior to use for immunoprecipitation. Proteins were either silver stained directly or transferred to Immobilon-P and revealed with India ink staining. A protein of about 116 kDa, detected by both silver stain (Fig. 1A) and India ink stain (Fig. 1B), could be reproducibly co-immunoprecipitated with CD45. When immunoprecipitates were prepared using M1/42, a monoclonal antibody of the same isotype as I3/2 but recognizing major histocompatibility complex class I molecules, no band at 116 kDa was observed (Fig. 1B). The 116-kDa protein could not be detected in immunoprecipitates prepared using anti-LFA-1 or anti-transferrin receptor as additional controls (data not shown). A 116-kDa protein is also detected in immunoprecipitates from CD45 SAKRTLS 12.1 cells but not CD45 SAKRTLS 12.1 following metabolic labeling with [S]methionine (Fig. 1C).


Figure 1:SDS-PAGE of CD45 immunoprecipitated from CD45 and CD45 cell lines. A, silver-stained gel of I3/2 immunoprecipitates prepared from 4 times 10^7 CD45 (+) and CD45 (-) SAKRTLS 12.1 cells. B, as in A except isotype-matched control antibody M1/42 was used in parallel and the proteins were detected by India ink staining of Immobilon-P after transfer. C, as in A except cells were metabolically labeled with [S]methionine. D, as in B except anti-CD45 antibody M1/9.34 HL2 was employed. Arrows indicate the position of p116.



To ascertain if the association of p116 with anti-CD45 immunoprecipitates is unique to the anti-CD45 monoclonal antibody that we employed, we have also used a second antibody to CD45, M1/9.34 HL2, and found that a 116-kDa protein is also present in these immunoprecipitates (Fig. 1D). Taken together, these results suggest that the 116-kDa protein specifically associates with CD45 and does not nonspecifically associate with the immunoprecipitating antibodies or the Sepharose beads. The association is most likely not due to cross-reactivity of the antibody because p116 is not immunoprecipitated from lysates of CD45 cells. Identical results have also been obtained using sets of CD45 and CD45 cell lines derived from NZB.1, BW5147, and Yac-1 T-lymphomas (data not shown).

p116 Is Associated with CD45 in a Number of Cell Types

We screened a number of different cell lines derived from different cell lineages to determine if p116 is associated with CD45 in all cells that express CD45. Since we do not have CD45 variants of all these different cell lines, we compared the I3/2 immunoprecipitates with parallel immunoprecipitates employing isotype-matched M1/42 as a negative control. The results of two such experiments using freshly isolated thymocytes from AKR mice and a cytotoxic T-cell clone called 2C are shown in Fig. 2A. It can be seen that a 116-kDa protein is found in the I3/2 immunoprecipitates but not in the M1/42 immunoprecipitates. In all, we have tested about 20 hematopoietic cell lines of different origins and have found, without exception, that all cell lines expressing CD45 have a 116-kDa protein present in CD45 immunoprecipitates. These cell lines include B and T tumor cell lines (A20.CY, LK35.2, 70Z/3, EL4, AKR), antigen-specific T-cell clones and hybridomas (clone 11, CTLL-2, L3, AODH 7.1, A1.1, DO.11.10), the mastocytoma P815, and the macrophage and monocytic cell lines WEHI-3, J774A.1 and P388D1 (data not shown).


Figure 2:CD45 immunoprecipitates from various cell types. A, CD45 (I3/2) or control (M1/42) immunoprecipitates prepared from freshly isolated C57Bl/6 thymocytes and from cytotoxic T-cell line 2C were detected by India ink staining after transfer of SDS-PAGE-separated proteins to Immobilon-P. B, India ink stain of I3/2 immunoprecipitates prepared from CD45 and CD45 SAKRTLS 12.1 as well as 2 cells expressing RO (2-CD45/O) or RB (2-CD45/BC) isoforms of CD45. Immunoprecipitates were also prepared from 2 control cells. All immunoprecipitates in B were washed 5 times in lysis buffer containing 0.5 M NaCl. Arrows denote the position of the 116-kDa protein.



We have obtained 2 cells that express various isoforms of CD45 that were generated by infection with recombinant retrovirus containing various isoforms of CD45(28) . As expected, we were unable to detect either CD45 or p116 in I3/2 immunoprecipitates from control 2 cells. Interestingly, we have found that a 116-kDa protein co-immunoprecipitates with two different isoforms of CD45 expressed in 2 cells (Fig. 2B). These results raise the possibility that p116 may be ubiquitously expressed, even in cells that do not normally express CD45. Furthermore, the association between CD45 and p116 appears to be relatively strong, in that it is stable in 0.5 M NaCl in 0.1-1% Nonidet P-40 or digitonin (Fig. 2B and data not shown).

p116 Is Associated with the External Domain and/or Membrane-proximal Region of CD45

A revertant of the BW5147/CD45 cell line that reexpresses CD45 has been isolated. The CD45 that is reexpressed, however, is truncated so that most of the cytoplasmic domain has been deleted as demonstrated by in vivo inorganic phosphate labeling studies(32) . It also lacks protein-tyrosine phosphatase enzymatic activity (4) but appears to express a normal external domain(32) . The exact location of this truncation has not yet been mapped; however, the CD45 that is expressed is about 120 kDa, which is consistent with it lacking essentially all of the cytoplasmic domain. We have found that p116 associates with CD45 expressed in this revertant cell line (Fig. 3, BW/rev). The amount of the 116-kDa protein found in an I3/2 immunoprecipitate from BW/rev is much lower than in the BW5147 parent, consistent with the revertant expressing about 10-20% of the CD45 expressed on parental BW5147 cells(4) . It is difficult to visualize the truncated CD45 band on India ink-stained membranes, but it does appear as a faint smear at about 120 kDa on silver-stained gels (data not shown). The observation that the 116-kDa protein is found in immunoprecipitates of this truncated CD45 suggests that it associates with the external domain, the transmembrane domain, and/or the membrane-proximal region of CD45.


Figure 3:CD45 immunoprecipitated from a cell line lacking most of the cytoplasmic domain. India ink staining of I3/2 immunoprecipitates prepared from the BW5147 parent (BW), a CD45 variant (BW/CD45) and a revertant (BW/rev) expressing a truncated cytoplasmic domain. The arrow indicates the position of p116.



p116 Is Not CD45 Isoform-restricted in Its Association

Since p116 appears to associate with CD45 through the external domain, the transmembrane domain, and/or the membrane-proximal region of the cytoplasmic domain, there is a formal possibility that the differentially expressed exons might be involved in these associations. To examine this possibility, we again made use of the 2 transfectants expressing various isoforms of CD45(28) . We examined four different CD45 cell lines expressing differentially spliced exons 4, 5, and 6 (RA), 5 and 6 (RB), 6 alone (RC) or none of the differentially spliced exons (RO). To perform the experiment, we prepared CD45 immunoprecipitates from each of these cell lines, including a control line, and mixed these with lysates prepared from [S]methionine-labeled CD45 SAKRTLS 12.1 cells to assay whether the labeled p116 from the CD45-negative cell line could associate with the unlabeled immunoprecipitated CD45. It can be seen in Fig. 4that p116 associates equally well with all of the different isoforms tested. These results suggest that the association between CD45 and p116 is isoform-independent and are consistent with our observation that the 116-kDa protein is detected in CD45 immunoprecipitates prepared from a variety of different cell types expressing different isoforms of CD45. Furthermore, these results also demonstrate that p116 is expressed in CD45 cells and can associate with CD45 in vitro.


Figure 4:CD45 immunoprecipitated from cell lines expressing various isoforms of CD45. I3/2 immunoprecipitates of SAKRTLS 12.1 CD45 and CD45 cells, control 2 cells, and 2 cells expressing CD45/C (RC), CD45/BC (RB), or CD45/ABC (RA). The immunoprecipitates were washed in lysis buffer and mixed with lysates from CD45 SAKRTLS 12.1 cells that had been metabolically labeled with [S]methionine. After a 60-min incubation at 4 °C, the immunoprecipitates were washed and subjected to SDS-PAGE followed by autoradiography. An arrow indicates the position of the 116-kDa protein.



Characterization of p116

To determine if p116 is a membrane-bound or cytosolic protein, we prepared membrane and cytosolic fractions from CD45 SAKRTLS 12.1 cells. The p116 expressed in these cells does not have CD45 to associate with and would therefore not be found in the membrane solely by virtue of its association with CD45. To perform this experiment, we immunopurified CD45 over an I3/2 affinity column. After this purification step, the CD45 appeared as a single band on a silver-stained gel (data not shown) and had no detectable p116 associated with it. The purified CD45 was mixed with I3/2 beads along with the Nonidet P-40-solubilized membranes or the cytosolic fraction isolated from CD45 SAKRTLS 12.1 cells. We also examined the detergent-solubilized membranes devoid of detergent-insoluble material. The beads were then washed and subjected to SDS-PAGE. The results of one such experiment are shown in Fig. 5. It can be seen that a protein of about 116 kDa becomes associated with CD45 after mixing with the Nonidet P-40-solubilized membrane fraction from CD45 cells but not with the cytosolic fraction, suggesting that p116 is a membrane-bound protein. The p116 is predominantly found in the Nonidet P-40-soluble membrane fraction further supporting its association with the membrane and not with the membrane skeleton. We were also able to reconstitute the association with postnuclear Nonidet P-40 extracts mixed with purified CD45 in the presence of I3/2 beads but not in the presence of control M1/42 beads (data not shown).


Figure 5:CD45 associates with a 116-kDa protein found in membranes isolated from CD45 cells. The cytosolic fraction (lanes3 and 4), Nonidet P-40-solubilized membrane fraction (lanes5 and 6), and Nonidet P-40-solubilized membrane fraction depleted of detergent-insoluble material (lanes7 and 8) prepared from CD45 SAKRTLS 12.1 cells were mixed with I3/2 beads in the presence (+) or absence (-) of purified CD45. Immune complexes were washed as described under ``Materials and Methods.'' As a control, beads and purified CD45 were incubated with lysis buffer rather than cell extracts (lanes1 and 2). I3/2 immunoprecipitates from 4 times 10^7 CD45 (+) and CD45(-) SAKRTLS 12.1 cells are also shown (lanes9 and 10). The arrow marks the position of p116.



To determine if the 116-kDa protein contains an external domain, we cell-surface biotinylated CD45 and CD45 SAKRTLS 12.1 cells and immunoprecipitated CD45 under conditions that maintain the association with p116. It is clear from Fig. 6A that p116 can be biotinylated, suggesting that it does have an external domain accessible to the biotinylating agent. Control immunoprecipitates containing p56 or actin demonstrate that intracellular molecules are not biotinylated (data not shown). Protein staining of parallel immunoprecipitates run on the same gel demonstrate that the biotinylated protein at 116 kDa comigrates with p116 as revealed by India ink staining and that both are found only in immunoprecipitates from CD45 cells (Fig. 6A). Interestingly, we also observe a 30-kDa biotinylated protein in immunoprecipitates prepared from CD45 cells but not CD45 cells. It is possible that this is the previously described 30-kDa adapter protein, which is predicted to have a very small external domain(26) . Surface iodination experiments have also been done, and p116 does become iodinated albeit to a very low level (data not shown). These results, in conjunction with the data presented in Fig. 5, suggest that p116 is a membrane-bound protein with an external domain.


Figure 6:p116 is a tyrosine-phosphorylated transmembrane glycoprotein. A, I3/2 immunoprecipitates were prepared in triplicate from CD45 (+) and CD45(-) SAKRTLS 12.1 cells following biotinylation of cell-surface proteins. Samples were loaded onto a single gel for SDS-PAGE and transferred to Immobilon-P for detection by India ink staining or blotting with either Streptavidin or anti-phosphotyrosine. An arrow indicates the position of the 116-kDa protein. B, I3/2 immunoprecipitates prepared from [S]methionine-labeled CD45 (lane1) or CD45 (lanes2 and 3) SAKRTLS 12.1 cells were treated with N-glycosidase F (+) or mock-treated(-) overnight at 37 °C. The immunoprecipitates were then washed and subjected to SDS-PAGE followed by fluorography. The arrowheads indicate the position of p116 with or without N-glycosidase F (EndoF) treatment.



Since CD45 is a protein-tyrosine phosphatase, the determination of the phosphorylation status of p116 is significant. We found, by anti-phosphotyrosine immunoblotting, that a CD45-associated 116-kDa protein is tyrosine phosphorylated (Fig. 6A). The immunoprecipitates used for immunoblotting were run on the same gel with a parallel immunoprecipitate that was stained with India ink to reveal p116. It is clear that the tyrosine-phosphorylated protein comigrates with p116 (Fig. 6A). Identical results were obtained with two different rabbit anti-phosphotyrosine antisera, and antibody binding to the 116-kDa protein could be inhibited with phenylphosphate or phosphotyrosine (data not shown). Furthermore, very little tyrosine phosphorylation is detected when sodium vanadate is omitted from the lysis buffer, suggesting that the phosphorylation of p116 is regulated by a tyrosine phosphatase (data not shown). Taken together, these results support the conclusion that p116 is a tyrosine-phosphorylated protein.

To determine if p116 is a glycoprotein, we incubated immunoprecipitates containing CD45 and p116 with endoglycosidase F/N-glycosidase F, which removes high mannose and complex N-glycans. As shown in Fig. 6B, there is a dramatic shift in the mobility of CD45, which serves as our internal control, and a slight shift in the mobility of p116, suggesting that p116 is a glycoprotein. When these immunoprecipitates are immunoblotted with anti-phosphotyrosine antibodies, there is an identical shift in the tyrosine phosphorylated protein, further supporting the conclusion that p116 is a tyrosine-phosphorylated and glycosylated protein (data not shown). It is also interesting to note that the association between CD45 and p116 is not dependent on N-linked glycosylation of either CD45 or p116 since the association is stable after N-glycosidase F treatment. These results taken together with the membrane reconstitution and surface biotinylation experiments suggest that p116 is a transmembrane glycoprotein.


DISCUSSION

We have found that a 116-kDa protein specifically associates with CD45 in all CD45-expressing cells examined. This molecule appears to be a tyrosine-phosphorylated transmembrane glycoprotein. The interaction between CD45 and this 116-kDa protein is relatively stable and is easily reconstituted. Chemical cross-linkers are not required to detect the association, which is stable in buffer containing Nonidet P-40 and 0.5 M NaCl. The association is stable overnight at 37 °C and does not require the presence of N-linked glycosylation (Fig. 6B).

A number of studies have been done to identify molecules that are associated with CD45 on the T-cell surface. It has been shown to be associated with Thy-1, the T-cell antigen receptor, CD2, CD4, and CD8 (20, 21, 22, 23) . The expression of these cell-surface molecules is restricted to the T-cell lineage and therefore does not permit generalizations about the role of CD45 in the large number of different cell types in which it is expressed. We have found that p116 is associated with CD45 in every hematopoietic cell type examined, including non-lymphoid cells. Intriguingly, p116 was also detected in cells that do not normally express CD45, suggesting that p116 is ubiquitously expressed.

It has been demonstrated that CD45 is found in CD26 (dipeptidyl peptidase IV) immunoprecipitates prepared from digitonin, but not Nonidet P-40, extracts of human T-lymphocytes(24) . This molecule is about 110 kDa and has a wide tissue distribution raising the possibility that it might be the equivalent of the 116-kDa protein described herein. However we find this unlikely for three reasons. 1) CD26 is efficiently iodinated, whereas p116 is minimally iodinated. 2) CD26 has a different mobility than p116 by SDS-PAGE. 3) Mouse CD26 has no tyrosine in the cytoplasmic domain for phosphorylation, and 4) antibodies to CD26 do not react with p116 either by immunoprecipitation or immunoblotting (data not shown).

A number of observations have allowed us to eliminate the possibility that p116 is a degradation product of CD45. First, neither antiserum to the cytoplasmic domain of CD45 nor monoclonal antibodies to the external domain react with p116 by Western blotting (data not shown). Second, CD45 cells express p116 as demonstrated by the labeled reconstitution experiments where labeled p116 from CD45 cells is able to bind to unlabeled CD45 (Fig. 4). Third, two-dimensional peptide mapping of CD45 versus p116 from [S]methioninelabeled cells gives very distinct patterns (data not shown). These observations, taken together, suggest that p116 is not a degradation product of CD45.

The identification of p116 will likely yield important functional information about CD45. We have worked out a purification scheme for p116 and are now trying to obtain sequence information. We have also immunized rabbits with the purified protein to obtain antiserum that will be useful for further characterization of the interaction. At present, one can only speculate about the potential functions for such a molecule. One possibility is that p116 regulates CD45 enzymatic activity either directly or by allowing or preventing interactions with its substrate(s). Since p116 is tyrosine phosphorylated, it could be a substrate of CD45 and therefore a downstream mediator of CD45-triggered signaling events. It is also possible that p116 may be a linker molecule that connects CD45 to additional signaling pathways similar to the -chain of the T-cell receptor complex(33, 34) . Consistent with this, p116 has limited glycosylation and is not readily iodinated, suggesting that it may have a small external domain as does the -chain(33) . Preliminary studies suggest that the phosphorylation of p116 does not change upon T-cell activation; however, this can be addressed more directly once we have antibodies to the protein. Further characterization of p116 will provide insight into the role of CD45 in signal transduction in lymphocytes and other hematopoietic cells.


FOOTNOTES

*
This work was supported in part by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the Leukemia Society of America. 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 by studentships from the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research.

To whom correspondence should be addressed. Tel.: 403-492-7710; Fax: 403-492-0368.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Drs. Pauline Johnson, Robert Hyman, and Jonathan Ashwell for the 2 transfectants, the CD45 and CD45 BW5147, NZB and SAKRTLS 12.1 cell lines, and the CD45 Yac-1 cells, respectively. We also thank Dr. Kevin Kane for a critical review of the manuscript.


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