Reconstitution of Interactions between Protein-tyrosine Phosphatase CD45 and Tyrosine-protein Kinase p56lck in Nonlymphoid Cells*

(Received for publication, September 11, 1996, and in revised form, February 28, 1997)

François G. Gervais Dagger § and André Veillette Dagger §par **Dagger Dagger §§

From the Dagger  McGill Cancer Centre and the Departments of § Biochemistry, par  Medicine, and ** Oncology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the Dagger Dagger  Departments of Medicine and Oncology, Montreal General Hospital, Montréal, Quebec H3G 1A4, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

To further understand the functional interactions between CD45 and p56lck in T-cells, we stably reconstituted their expression in a nonlymphoid system. The results of our analyses demonstrated that CD45 could dephosphorylate tyrosine 505 of p56lck in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56lck interfered with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56lck. The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells was also greatly influenced by the catalytic activity of p56lck. Indeed, whereas CD45 provoked dephosphorylation of kinase-defective Lck molecules in this system, it failed to stably dephosphorylate kinase-active p56lck polypeptides. Finally, our studies showed that CD45 was also able to inhibit the oncogenic potential of a constitutively activated version of p56lck in NIH 3T3 cells. This effect did not require the Lck unique domain and apparently resulted from selective dephosphorylation of substrates of activated p56lck in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56lck in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56lck-mediated functions in vivo.


INTRODUCTION

p56lck is a lymphocyte-specific member of the Src family of tyrosine-protein kinases (Refs. 1-3; reviewed in Refs. 4 and 5). Like the other members of the Src family, it bears several independent structural domains that include, from the amino terminus to the carboxyl terminus: (i) sites of myristylation (glycine 2) and palmitylation (cysteines 3 and/or 5), involved in targetting to cellular membranes (Ref. 6; reviewed in Ref. 7); (ii) a unique domain of roughly 50 amino acids, which mediates binding to the CD4 and CD8 T-cell surface antigens (8-12); (iii) Src homology (SH)1 3 and SH2 domains, capable of interacting with proline-rich motifs and phosphotyrosine-containing sequences, respectively (reviewed in Ref. 13); (iv) a kinase domain, including sites for ATP-binding, phosphotransfer (lysine 273) and autophosphorylation (tyrosine 394) and (v) a negative regulatory domain, encompassing the major site of in vivo tyrosine phosphorylation, tyrosine 505.

The catalytic activity of p56lck is primarily regulated by phosphorylation of tyrosines 394 and 505 (reviewed in Ref. 5). Phosphorylation at tyrosine 394 activates the catalytic function of p56lck, by provoking a conformational change in the kinase domain (14). Conversely, phosphorylation at tyrosine 505 inhibits the enzymatic activity of Lck (15-17), presumably by allowing an intramolecular interaction between the carboxyl-terminal end and the SH2 domain of the enzyme. Accumulating data indicate that tyrosine 505 phosphorylation is mediated by p50csk, a tyrosine-protein kinase expressed in all cell types (17-19; reviewed in Ref. 20). However, tyrosine 505 can also be a site of autophosphorylation. This possibility is suggested by the findings that p56lck could undergo phosphorylation at tyrosine 505 in bacteria (which lack endogenous tyrosine-protein kinases) or during in vitro kinase reactions (21, 22). Nonetheless, autophosphorylation may not be a prominent component of tyrosine 505 phosphorylation in mammalian cells, because a kinase-defective version of p56lck (lysine 273 to arginine 273 (Arg273) Lck) was still extensively phosphorylated at this site in transfected NIH 3T3 fibroblasts (19).

Contrary to Lck polypeptides expressed in NIH 3T3 cells, those isolated from T-lymphocytes are poorly phosphorylated at tyrosine 505 (23). This difference is seemingly consequent to the action of CD45, a transmembrane protein-tyrosine phosphatase selectively expressed in nucleated hemopoietic cells (for a review, see Ref. 24). This notion is supported by the observation that Lck polypeptides immunoprecipitated from CD45-deficient T-cells exhibited a marked (8-10-fold) increase in tyrosine 505 occupancy, when compared with p56lck molecules recovered from their CD45-positive counterparts (25-29). In contrast, expression of CD45 had little or no effect on the tyrosine phosphorylation of two other Src family kinases, p59fynT and p60c-src (26). The lack of constitutive dephosphorylation of tyrosine 505 in CD45-negative T-cells is thought to explain the inability of these cells to become activated upon stimulation with antigen or anti-T-cell receptor antibodies (30-33).

Over the past few years, significant efforts have been directed toward understanding the mechanism(s) by which CD45 causes selective dephosphorylation of p56lck in T-cells. Because a small fraction of Lck molecules can be co-immunoprecipitated with CD45 in mild detergent lysates of T-cells (34, 35), it is likely that these two molecules are in close proximity in the cell. Interestingly, we demonstrated that deletion of the unique domain of p56lck or its replacement by the equivalent sequence from another Src family kinase (p59fynT) significantly reduced dephosphorylation at tyrosine 505 in a CD45-positive T-cell line (23). This finding implied that the unique domain contains elements that favor the functional interactions between CD45 and p56lck. Because the unique domain of p56lck can associate with the cytoplasmic portion of CD45 in vitro (36), it is conceivable that Lck directly associates with CD45 in vivo via these sequences. However, it is also plausible that other molecules participate in this interaction. One proposed candidate is the CD45-associated protein, a hemopoietic cell-specific polypeptide having the capacity to bind both CD45 and p56lck (37-40).


MATERIALS AND METHODS

Cells

NIH 3T3 mouse fibroblasts and psi -2 packaging cells were grown in alpha -minimal essential medium (41) containing 10% fetal calf serum and antibiotics. Derivatives expressing the neomycin phosphotransferase were grown in the additional presence of G418 (0.5 mg/ml), whereas cells expressing the puromycin resistance gene were propagated in medium supplemented with puromycin (1 µg/ml).

Antibodies

Anti-mouse CD45 rat monoclonal antibody (mAb) M1.89.18.7 was obtained from the American Type Culture Collection. Purified antibodies were used in our experiments. Rabbit anti-CD45 polyclonal antibodies were generated against the carboxyl-terminal tail of CD45 and were kindly provided by Dr. Phil Branton (McGill University, Montreal, Quebec, Canada). Rabbit antisera directed against amino acids 2-148 of p56lck (42) or against the SH3 domain of p56lck (23) were reported previously. Rabbit antisera directed against annexin II and GTPase-activating protein (GAP) of p21ras were generously provided by Drs. Tony Hunter (The Salk Institute, La Jolla, CA) and Tony Pawson (Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada), respectively. Anti-cortactin mAbs were kindly provided by Dr. Tom Parsons (University of Virginia, Charlottesville, VA). Affinity-purified rabbit anti-phosphotyrosine antibodies were generated in our laboratory,2 whereas anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY).

Expression of CD45 in NIH 3T3 Cells

A cDNA encoding the R0 (T200) isoform of mouse CD45 (cloned in the expression vector pHbeta Apr-1-neo) was kindly provided by Dr. Pauline Johnson (University of British Columbia, Vancouver, British Columbia, Canada). This construct was stably introduced in NIH 3T3 cells by calcium phosphate precipitation (43). Monoclonal G418-resistant cell lines were established by limiting dilution and screened by fluorescence-activated cell sorter (FACS) analysis using mAb M1.89.18.7 and fluorescein isothiocyanate-conjugated goat anti-rat IgG.

Expression of Lck Polypeptides in NIH 3T3 Cells

cDNAs encoding wild-type p56lck, lysine 273 to arginine 273 (Arg273) p56lck, an Arg273 Lck variant lacking amino acids 16-62 of the unique domain (Delta 16-62R273 Lck), or tyrosine 505 to phenylalanine 505 (Phe505) Lck were described elsewhere (15, 23). An lck cDNA containing the Phe505 mutation, in addition to a deletion of amino acids 16-62, was created by standard recombinant DNA technology. These cDNAs were inserted in the multiple cloning site of the retroviral expression vector pBabePuro (44), which also contains the puromycin resistance gene (puro). The retroviral constructs were stably transfected in psi -2 packaging cells by calcium phosphate precipitation (43), and retroviral supernatants were used to infect NIH 3T3 cells expressing either CD45 or the neomycin phosphotransferase alone. Cells expressing the various Lck polypeptides were selected by growth in puromycin-containing medium in the continuous presence of G418.

Immunoprecipitations, Immunoblots, and Peptide Mapping

Immunoprecipitations and immunoblots were conducted as described elsewhere (8), with the exception of CD45 immunoprecipitations, which were performed using mAb M1.89.18.7 (10 µg) coupled to protein G-Sepharose (Pharmacia Biotech Inc.). Following immunoblotting, immunoreactive products were detected by autoradiography and quantitated using a PhosphorImager (BAS 2000, Fuji). Peptide mapping with cyanogen bromide was also performed as reported previously (19).

Pervanadate Treatment

To inhibit protein-tyrosine phosphatase activity, cells were treated for 10 min at 37 °C with the protein-tyrosine phosphatase inhibitor pervanadate (1:50, v/v) as described elsewhere (19). Lck polypeptides were recovered by immunoprecipitation and subjected to further analyses as outlined above.

Cell Fractionation

Cell fractionation studies were conducted according to a previously described protocol (23).

Cell Transformation Assays

To examine focus formation, NIH 3T3 derivatives (103 or 104 cells) were seeded in 6-well Costar plates with 105 neomycin-resistant NIH 3T3 cells. Foci were counted after 9 days of growth. For growth in soft agar, 2 × 104 cells were plated in medium containing 0.3% agar as described previously (15). Colony formation was monitored for 2 weeks. All assays were done in duplicate and were repeated at least three times.


RESULTS

Generation of CD45-positive Variants of NIH 3T3 Fibroblasts

To further dissect the regulation of p56lck by CD45 in T-cells, we wished to recreate the expression of these two molecules in a nonlymphoid mammalian cell system. To this end, mouse NIH 3T3 fibroblasts were chosen, because they do not normally express either polypeptide. As a first step, NIH 3T3 variants expressing the isoform of CD45 predominantly contained in mature T-cells, CD45 R0/T200 (24), were generated as outlined under "Materials and Methods." Two CD45-positive NIH 3T3 clones (clones 2 and 31) were selected for further studies. As depicted in Fig. 1 (A and B), FACS analyses showed that these two cell lines expressed easily appreciable amounts of CD45 at their surface. To ensure that full-length CD45 polypeptides were expressed in these cells, CD45 was immunoprecipitated with mAb M1.89.18.7 and subsequently immunoblotted with a polyclonal rabbit anti-CD45 serum (Fig. 1C). This experiment revealed that the two clones contained a ~180-kDa immunoreactive polypeptide (lanes 2 and 3) that comigrated with the CD45 molecule immunoprecipitated from the mouse T-cell line BI-141 (lane 4). The CD45-positive fibroblasts expressed approximately 10 times lower amounts of CD45 than BI-141 T-cells.


Fig. 1. Expression of CD45 in NIH 3T3 cells. A and B, FACS analyses. CD45-positive clones 2 (A) and 31 (B) were stained with either anti-CD45 mAb M1.89.18.7 (dashed line) or no antibody (solid line), followed by fluorescein isothiocyanate-conjugated goat anti-rat IgG. Immunofluorescence was detected by FACS analysis. In panel A, neomycin-resistant control NIH 3T3 cells were also stained with anti-CD45 mAb (dotted line). C, anti-CD45 immunoblot. Cells were lysed in nonionic detergent-containing buffer, and CD45 was immunoprecipitated (I.P.) from 1 mg of total cell proteins using anti-CD45 mAb M1.89.18.7. CD45 expression was then measured by immunoblotting with a polyclonal rabbit anti-CD45 serum. The position of CD45 is shown on the left, and those of prestained molecular mass markers (in kDa) are shown on the right. Exposure was 15 h.
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CD45 Can Induce Dephosphorylation of p56lck in NIH 3T3 Cells

Next, CD45-positive and CD45-negative NIH 3T3 cells were infected with retroviruses encoding either wild-type or kinase-defective (Arg273) p56lck molecules. Kinase-defective Lck molecules were included in these studies to eliminate the possibility of compensatory autophosphorylation at tyrosine 505, as discussed elsewhere (19, 23). Although the results reported herein primarily concerned CD45-positive clone 31, similar results were obtained with clone 2 (data not shown).

The impact of CD45 on the tyrosine phosphorylation of p56lck in NIH 3T3 cells was first examined (Fig. 2). Lck polypeptides were recovered by immunoprecipitation, and their phosphotyrosine content was assessed by immunoblotting with anti-phosphotyrosine mAb 4G10 (Fig. 2A, top panel). Moreover, the abundance of p56lck was verified by immunoblotting of parallel anti-Lck immunoprecipitates with an antiserum generated against the Lck SH3 region (middle panel). This was taken into consideration when determining the relative extents of Lck tyrosine phosphorylation in these various cell lines. Finally, the levels of CD45 were monitored by immunoblotting of anti-CD45 immunoprecipitates with rabbit anti-CD45 antibodies (bottom panel).


Fig. 2. Effects of CD45 expression on p56lck tyrosine phosphorylation in NIH 3T3 cells. A, anti-phosphotyrosine immunoblot. The extent of tyrosine phosphorylation of either wild-type (W) or kinase-inactive Arg273 (R) Lck polypeptides in the presence (+) or the absence (-) of CD45 was ascertained by anti-phosphotyrosine (alpha -P-tyr) immunoblotting of Lck immunoprecipitates (top). The abundance of Lck was measured by immunoblotting of parallel immunoprecipitates with an anti-Lck serum (middle), whereas that of CD45 was verified by immunoblotting of anti-CD45 immunoprecipitates with a rabbit anti-CD45 serum (bottom). The positions of Lck and CD45 are indicated on the left. Exposures were 36 (top), 4 (middle panel), and 36 h (bottom panel). I.P., immunoprecipitation. B, peptide mapping studies. Phosphorylation sites were evaluated by peptide mapping of 32Pi-labeled p56lck using cyanogen bromide. The position of the C3 fragment (which bears tyrosine 505) is shown on the left. The migrations of prestained molecular mass markers are indicated in kDa on the right. Exposure was 2 days.
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In keeping with our previous report (19), wild-type p56lck (Fig. 2A, lane 2) and Arg273 p56lck (lane 3) were tyrosine phosphorylated to similar extents in NIH 3T3 cells lacking CD45. However, although tyrosine phosphorylation of wild-type p56lck was not decreased by expression of CD45 (lane 4), the phosphotyrosine content of kinase-inactive Lck molecules was reduced ~5-fold (lane 5). The differential impact of CD45 on wild-type and Arg273 p56lck was not caused by variations in the expression levels of either Lck or CD45, because the two cell lines contained comparable quantities of these two products (middle and bottom panels).

To identify the sites of tyrosine phosphorylation on these Lck molecules, peptide mapping studies were conducted using cyanogen bromide. Cells were metabolically labeled with 32Pi, and Lck polypeptides were recovered by immunoprecipitation. Following cleavage with cyanogen bromide, phosphorylated peptides were resolved by gel electrophoresis and detected by autoradiography (Fig. 2B). Great care was taken to ensure that cleavage of p56lck by cyanogen bromide was complete and that equivalent amounts of Lck peptides were applied in each lane.

Cleavage of p56lck with cyanogen bromide results in three possible phosphopeptides: C1 (28 kDa), which contains potential amino-terminal sites of serine, threonine, and tyrosine phosphorylation; C2 (10 kDa), which bears tyrosine 394, the major site of in vitro autophosphorylation; and C3 (4 kDa), which carries tyrosine 505, the major site of in vivo tyrosine phosphorylation (45-47). In CD45-negative fibroblasts, both wild-type (lane 1) and kinase-defective (lane 2) p56lck were extensively phosphorylated within the tyrosine 505-containing C3 fragment, in agreement with earlier reports (19). As expected from the anti-phosphotyrosine immunoblot (Fig. 2A), expression of CD45 did not noticeably influence the extent of tyrosine 505 phosphorylation of wild-type Lck (Fig. 2B, lane 3). In contrast though, it caused a marked reduction of carboxyl-terminal phosphorylation of Arg273 Lck (lane 4). Although phosphorylation within the amino-terminal C1 fragment is not observed in Fig. 2B, it could be seen in longer autoradiographic exposures of this gel. Phosphorylation of the C1 peptide was not affected by CD45 expression (data not shown).

To verify that CD45 reduced the phosphorylation of kinase-inactive Lck through its phosphatase activity and not through other mechanisms such as steric hindrance, we tested the influence of pervanadate, a potent protein-tyrosine phosphatase inhibitor (Fig. 3). Cells were treated with pervanadate for 10 min and then processed for anti-Lck immunoprecipitations as described above. Anti-phosphotyrosine immunoblotting of these immunoprecipitates demonstrated that the protein-tyrosine phosphatase inhibitor caused a marked (~8-fold) increase in the phosphotyrosine content of Arg273 Lck in CD45-positive NIH 3T3 cells (Fig. 3A, compare lanes 2 and 4). Cyanogen bromide cleavage analysis (Fig. 3B) established that pervanadate restored tyrosine 505 phosphate occupancy of Arg273 Lck (lane 4). In addition, the phosphatase inhibitor provoked phosphorylation of both wild-type (lane 3) and kinase-defective (lane 4) p56lck within the tyrosine 394-containing C2 fragment, in keeping with earlier studies (19, 48, 49). Although the basis for phosphorylation of kinase-inactive Lck molecules at the "autophosphorylation" site has not yet been elucidated, this finding suggests that another cellular tyrosine-protein kinase can transphosphorylate tyrosine 394 in vivo (19, 48, 49).


Fig. 3. Effects of the protein-tyrosine phosphatase inhibitor pervanadate on Lck tyrosine phosphorylation in CD45-positive NIH 3T3 cells. A, anti-phosphotyrosine immunoblot. CD45-positive NIH 3T3 cell lines expressing either wild-type (W) or kinase-inactive Arg273 (R) Lck were incubated for 10 min at 37 °C in the presence (+) or the absence (-) of pervanadate (PV). Lck tyrosine phosphorylation was monitored as outlined in the legend of Fig. 2A. The position of p56lck is shown on the left. Exposure was 22 h. I.P., immunoprecipitation. B, peptide mapping studies. Sites of phosphorylation were defined by cyanogen bromide cleavage analyses as described in the legend of Fig. 2B. The positions of C1 (which contains amino-terminal sites of serine, threonine, and tyrosine phosphorylation), C2 (which encompasses tyrosine 394), and C3 (which bears tyrosine 505) are shown on the left. The migrations of prestained molecular mass markers are indicated in kDa on the right. Exposure was 15 h.
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The Unique Domain of p56lck Is Necessary for CD45-mediated Dephosphorylation of Tyrosine 505 in NIH 3T3 Cells

To verify that dephosphorylation of tyrosine 505 in NIH 3T3 cells occurred via a mechanism similar to that existing in T-cells, we examined whether the unique domain of p56lck was also necessary for the effect of CD45 in fibroblasts. Arg273 Lck polypeptides lacking residues 16-62 (Delta 16-62R273 Lck) were introduced in CD45-negative and CD45-positive NIH 3T3 cells. Anti-phosphotyrosine immunoblotting of anti-Lck immunoprecipitates (Fig. 4A, top panel) established that Delta 16-62R273 Lck (lane 3) was tyrosine phosphorylated to nearly the same extent as Arg273 p56lck (lane 1) in CD45-negative cells. In cells expressing CD45, however, the tyrosine phosphorylation of Delta 16-62R273 Lck (lane 4) was only reduced ~1.5-fold, in comparison with the ~5-fold decrease noted above for Arg273 Lck (lane 2; Fig. 2A, lanes 3 and 5). Complementary peptide mapping analyses confirmed that, unlike Arg273 Lck (Fig. 4B, lane 2), Delta 16-62R273 Lck remained prominently phosphorylated at tyrosine 505 in CD45-positive cells (lane 4).


Fig. 4. Impact of deletion of the unique domain on Lck tyrosine phosphorylation in NIH 3T3 cells. A, anti-phosphotyrosine immunoblot. NIH 3T3 cell lines expressing either Arg273 (R) or Delta 16-62R273 (Delta U-R) Lck in the presence (+) or the absence (-) of CD45 were lysed in nonionic detergent-containing buffer. The extent of Lck tyrosine phosphorylation was monitored by anti-phosphotyrosine immunoblotting of anti-Lck immunoprecipitates (top panel). The abundance of Lck was also determined by immunoblotting of parallel immunoprecipitates with an antiserum directed against the SH3 domain of p56lck (bottom panel), whereas the levels of CD45 were verified by immunoblotting of anti-CD45 immunoprecipitates with a rabbit anti-CD45 serum. The positions of Lck and CD45 are indicated on the left. Exposures were 28 (top panel) and 15 h (middle and bottom panels). B, peptide mapping studies. Sites of phosphorylation were defined by cyanogen bromide cleavage analyses as described in the legend of Fig. 2B. The position of the C3 fragment, which encompasses tyrosine 505, is shown on the left. The migrations of prestained molecular mass markers are indicated in kDa on the right. Exposure was 2 days. C, cell fractionation studies. Cell were mechanically lysed in hypotonic buffer, and lysates were separated into particulate (P) and cytosolic (S) fractions by differential centrifugation. After extraction in detergent-containing buffer, Lck polypeptides were recovered by immunoprecipitation (I.P.) and detected by immunoblotting. The positions of Lck are shown on the left. Exposure was 13 h.
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Because CD45 is a membrane-bound phosphatase, we wished to ensure that the association of p56lck with membranes in NIH 3T3 cells was not altered by deletion of the unique region. After incubation in hypotonic buffer, cells were mechanically broken in a Dounce homogenizer. Lysates were then fractionated by differential centrifugation, and Lck polypeptides were recovered from the particulate (P100) and cytosolic (S100) fractions by immunoprecipitation. The abundance of Lck in each fraction was determined by anti-Lck immunoblotting. These studies demonstrated that as is the case for Arg273 p56lck (Fig 4C, lanes 1 and 3), more than 80% of Delta 16-62R273 Lck was localized to the P100 fraction of either CD45-negative (lane 5) or CD45-positive (lane 7) NIH 3T3 cells.

CD45 Inhibits the Oncogenic Potential of an Activated Version of p56lck in NIH 3T3 Cells

Introduction of a constitutively activated version of p56lck (tyrosine 505 to phenylalanine 505 p56lck) in NIH 3T3 cells causes marked tyrosine phosphorylation of several intracellular proteins (15, 16). This biochemical modification leads to oncogenic cellular transformation, with its characteristic morphological alterations and the ability to form foci in monolayers and grow in semi-solid medium. To test whether CD45 could also regulate activated p56lck molecules, CD45-positive NIH 3T3 cells were infected with retroviruses encoding Phe505 p56lck. CD45-negative neomycin-resistant NIH 3T3 cells (Neo) were used as control recipient. Immunoblot analyses showed that all infected cells expressed comparable amounts of Phe505 p56lck (data not shown; see Fig. 7A, bottom panel).


Fig. 7. Impact of CD45 on tyrosine phosphorylation of Phe505 p56lck in NIH 3T3 cells. A, anti-phosphotyrosine immunoblot. As described in the legend of Fig. 2A, the position of p56lck is indicated on the left. Exposure was 28 h. I.P., immunoprecipitations. B, peptide mapping studies. As described in the legend of Fig. 2B, the migrations of the C1 and C2 peptides are indicated on the left, whereas those of prestained molecular mass markers in kDa are shown on the right. Exposure was 2 days.
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Whereas fibroblasts expressing either the neomycin phosphotranferase alone (Fig. 5A) or CD45 alone (Fig. 5B) were flat and possessed short plasma membrane extensions, those containing Phe505 p56lck without CD45 (Fig. 5C) were rounded and refractile and displayed multiple neuronal-like processes (15, 16). By contrast, cells expressing Phe505 p56lck and CD45 (Fig. 5D) exhibited an intermediate morphology, being less rounded, less refractile, and showing fewer processes than cells containing activated Lck alone (Fig. 5C). The ability of these cells to form foci in monolayer cultures and colonies in soft agar was also assessed (Table I). Like cells containing Phe505 Lck alone, cells expressing Phe505 Lck and CD45 could form foci in monolayers. However, the foci were ~five times smaller than those generated by Phe505 Lck-expressing cells. Furthermore, although fibroblasts expressing Phe505 p56lck formed large colonies in soft agar, cells expressing Phe505 Lck and CD45 failed to grow under this condition.


Fig. 5. Morphology of NIH 3T3 derivatives. Cells were examined by light microscopy. Magnification, 200×.
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Table I. Biological characteristics of NIH 3T3 derivatives


Cell line Focus formation Growth in soft agar Morphologya

% %
Experiment 1 
  Neo 0 0  -
  CD45+ clone 31 0 0  -
  Phe505 p56lck 18.7b 13.7 +++
  CD45+ clone 31 + Phe505 p56lck 12.0c 0 +
Experiment 2 
  CD45+ clone 2 + Arg273 p56lck NDd 0  -
  Phe505 p56lck ND d 12.1 +++
  CD45+ clone 2 + Phe505p56lck ND d 0 +

a -, similar to parental NIH 3T3 cells; +, some rounding up with few visible cellular processes; +++, rounded, refractile, with multiple neuronal-like processes.
b Foci of 1.5-2.0 mm.
c Foci of 0.3-0.4 mm.
d ND, not determined.

To elucidate the mechanism by which CD45 prevented oncogenic transformation by Phe505 p56lck, we studied its impact on the accumulation of phosphotyrosine-containing proteins (Fig. 6). Anti-phosphotyrosine immunoblotting of total cell lysates demonstrated that cells expressing Phe505 Lck and CD45 (Fig. 6A, lane 4) contained lower amounts of phosphotyrosine-containing proteins than cells expressing Phe505 p56lck alone (lane 3). This diminution especially affected polypeptides of 140, 120, and 36 kDa. Individual substrates were also recovered by immunoprecipitation with specific antibodies, and their phosphotyrosine content was determined by anti-phosphotyrosine immunoblotting (Figs. 6, B-D). In keeping with the analysis of total cell lysates (Fig. 6A), tyrosine phosphorylation of the 36-kDa annexin II was absent in cells expressing Phe505 Lck and CD45 (Fig. 6B, lane 4) in contrast to cells containing Phe505 Lck alone (lane 3). However, CD45 had little or no impact on the extent of tyrosine phosphorylation of cortactin (Fig. 6C), as well as on that of GAP and its associated p190 and p62 (Fig. 6D). We were not able to determine the identity of the 140- and 120-kDa substrates that appeared to be tightly regulated by CD45 in Phe505 p56lck-expressing cells (Fig. 6A, lane 4).


Fig. 6. Effect of CD45 on tyrosine protein phosphorylation in Phe505 p56lck-expressing NIH 3T3 cells. A, total cell lysates. Levels of intracellular tyrosine protein phosphorylation were determined by anti-phosphotyrosine immunoblotting of total cell lysates. The positions of the predominant substrates are indicated on the left, whereas those of prestained molecular mass markers are shown in kDa on the right. Exposure was 3 h. B, annexin II immunoprecipitations. Annexin II was immunoprecipitated from equivalent amounts of cell lysates with anti-annexin II antibodies. Its phosphotyrosine content was subsequently determined by anti-phosphotyrosine immunoblotting (top panel), whereas its abundance was ascertained by reprobing the stripped membrane with anti-annexin II antibodies (bottom panel). The position of annexin II is shown on the left, whereas that of a prestained molecular mass marker in kDa is indicated on the right. Exposures were 3 (top panel) and 2 days (bottom panel). C, cortactin immunoprecipitations (I.P.). Cortactin was immunoprecipitated with anti-cortactin antibodies, and its phosphotyrosine content determined by anti-phosphotyrosine immunoblotting (top panel). The abundance of cortactin was evaluated by reprobing the stripped membrane with anti-cortactin antibodies (bottom panel). The position of cortactin is shown on the left, whereas that of a prestained molecular mass marker in kDa is indicated on the right. Exposures were 3 (top panel) and 2 h (bottom panel). D, GAP immunoprecipitations. GAP was immunoprecipitated with anti-GAP antibodies. Its phosphotyrosine content, as well as that of the GAP-associated p190 and p62, was determined by anti-phosphotyrosine immunoblotting (top panel). The abundance of GAP was assessed by reprobing the stripped membrane with anti-GAP antibodies (bottom panel). The positions of GAP and its associated proteins are shown on the left, whereas those of prestained molecular mass markers in kDa are indicated on the right. Exposures were 16 (top panel) and 13 h (bottom panel).
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We wanted to determine whether the effect of CD45 on Phe505 Lck-expressing cells was due to dephosphorylation of Phe505 p56lck or to dephosphorylation of downstream targets. To this end, the phosphotyrosine content of Phe505 Lck was first examined by anti-phosphotyrosine immunoblotting of anti-Lck immunoprecipitates (Fig. 7A). This study showed that expression of CD45 reduced the extent of tyrosine phosphorylation of Phe505 p56lck by ~2-fold. To ascertain whether these changes were due to dephosphorylation of tyrosine 394, the positive regulatory site of p56lck, peptide mapping studies were conducted (Fig. 7B). These analyses failed to show any reduction in phosphorylation of the C2 fragment of p56lck, which contains tyrosine 394. Although a small decrease in C1 phosphorylation could be seen in CD45-positive cells in this experiment (Fig. 7B, lane 2), a similar change was not observed in other experiments (data not shown).

These findings raised the possibility that CD45 did not act by dephosphorylating Phe505 p56lck but rather by dephosphorylating its substrates. To help support this idea, the impact of CD45 on transformation by a variant of Phe505 Lck lacking the unique domain was tested (Fig. 8 and Table II). Because the unique domain plays an important role in the CD45-mediated dephosphorylation of p56lck (this report and Ref. 23), we reasoned that removal of this domain should have no impact on the effect of CD45 if it were due to dephosphorylation of downstream substrates. Thus, CD45-positive and CD45-negative NIH 3T3 cells were infected with retroviruses encoding Delta 16-62F505 Lck. Cells containing Delta 16-62F505 Lck without CD45 were morphologically transformed in a manner analogous to Phe505 Lck-expressing cells (Table II and data not shown). Moreover, these cells were capable of growing in soft agar, albeit with a slightly lower efficiency than Phe505 Lck-expressing cells (Table II). Anti-phosphotyrosine immunoblotting of total cell lysates showed that Delta 16-62F505 Lck-expressing cells also possessed elevated levels of phosphotyrosine-containing proteins (Fig. 8, lane 5).


Fig. 8. Effect of CD45 on tyrosine protein phosphorylation in Delta 16-62F505 Lck-expressing NIH 3T3 cells. Total cell lysates were probed by immunoblotting with either anti-phosphotyrosine antibodies (top panel) or antibodies directed against the SH3 region of Lck (bottom panel). The positions of the major tyrosine phosphorylation substrates as well as of Lck are indicated on the left. Those of prestained molecular mass markers are shown on the right in kDa. Exposures were 3 (top panel) and 24 h (bottom panel).
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Table II. Biological characteristics of NIH 3T3 derivatives


Cell line Growth in soft agar Morphologya

%
Neo 0 0
CD45+ clone 31 0 0
Phe505 p56lck 16.4b +++
CD45+ clone 31 + Phe505 p56lck 0 +
 Delta 16-62F505 Lck 10.4c +++
CD45+ clone 31 + Delta 16-62 F505 Lck 0 +

a -, similar to parental NIH 3T3 cells; +, some rounding up with few visible cellular processes; +++, rounded, refractile, with multiple neuronal-like processes.
b Colonies of 0.5-1.0 mm.
c Colonies of 0.2-0.3 mm.

As was the case for cells containing Phe505 Lck and CD45, cells expressing Delta 16-62F505 Lck and CD45 exhibited a morphology that was intermediate between those of Neo cells and Delta 16-62F505 Lck-expressing cells (Table II and data not shown). Furthermore, they were unable to grow in soft agar (Table II). CD45 also reduced the accumulation of phosphotyrosine-containing proteins in Delta 16-62F505 Lck-expressing cells (Fig. 8, lane 6) in manner similar to that observed in cells expressing Phe505 p56lck (lane 4). Hence, these data indicated that CD45 inhibited Phe505 Lck-mediated transformation by a process independent of the Lck domain.


DISCUSSION

Herein, we report the first successful attempt at reconstituting the CD45-mediated regulation of p56lck in a nonlymphoid system. The results of our experiments showed that expression of the R0 isoform of CD45 in NIH 3T3 fibroblasts caused a ~5-fold decrease in the extent of tyrosine 505 phosphorylation of kinase-defective (Arg273) p56lck molecules. This effect was most likely due to the phosphatase activity of CD45, because treatment with pervanadate, a protein-tyrosine phosphatase inhibitor, restored phosphorylation of the carboxyl-terminal tyrosine of Lck in CD45-positive NIH 3T3 cells. As demonstrated for BI-141 T-cells (23), the unique region of p56lck was also found to play an important role in the CD45-induced dephosphorylation of tyrosine 505 in NIH 3T3 cells. Thus, dephosphorylation of the inhibitory tyrosine of p56lck by CD45 can occur in a nonlymphoid cellular system by a mechanism similar to that taking place in T-cells.

These findings implied that no other lymphoid-specific components are absolutely necessary for the CD45-mediated dephosphorylation of p56lck. Specifically, they also demonstrated that CD45-associated protein, a protein that can associate with both CD45 and p56lck (37-41) and is not expressed in NIH 3T3 cells,2 is not critical for the action of CD45 on p56lck. This idea was further supported by our finding that enforced expression of CD45-associated protein in NIH 3T3 cells did not modify the ability of CD45 to dephosphorylate wild-type, Arg273, or Phe505 Lck molecules.2 Nevertheless, it is likely that additional features or processes enhance the ability of CD45 to dephosphorylate tyrosine 505 in T-cells. Indeed, although CD45 provoked dephosphorylation of kinase-inactive Lck molecules in fibroblasts, it failed to stably dephosphorylate wild-type Lck polypeptides. In contrast, the two forms of Lck were equally well dephosphorylated in T-cells (23). The higher amounts of CD45 typically present in T-cells may explain this difference. Possibly, greater amounts of CD45 are needed for stable dephosphorylation of kinase-active Lck polypeptides in vivo. Alternatively, the presence of other yet unidentified molecules interacting with CD45, Lck or both, may contribute to more efficient dephosphorylation of Lck in T-cells.

These findings provided clear evidence that the catalytic activity of p56lck can influence the ability of CD45 to stably dephosphorylate tyrosine 505. It is conceivable that the enzymatic function of p56lck antagonizes the effect of CD45 by allowing autophosphorylation at tyrosine 505 (21, 22). Alternatively, the enzymatic activity of Lck may facilitate the recruitment of p50csk, the tyrosine-protein kinase normally responsible for phosphorylating tyrosine 505 (Refs. 17-19). Lastly, it is possible that active Lck molecules can modify the function of CD45 and reduce its ability to dephosphorylate tyrosine 505. Regardless of the mechanism underlying this phenomenon, these observations raised the interesting possibility that activation of p56lck may diminish the capacity of CD45 to dephosphorylate tyrosine 505 in T-cells, thereby providing a potential negative feedback mechanism. This concept may explain the earlier finding that CD4-mediated activation of p56lck in T-cells led to a paradoxical augmentation of tyrosine 505 phosphorylation, in addition to the expected increase in tyrosine 394 phosphorylation (50, 51).

The impact of CD45 on an activated version of p56lck (Phe505 Lck) was also evaluated. Contrary to cells containing Phe505 p56lck alone, cells harboring Phe505 p56lck and CD45 exhibited a less transformed morphology, formed smaller foci in monolayer cultures, and were unable to grow in soft agar. These biological effects were accompanied by a noticeable reduction in the extent of tyrosine protein phosphorylation induced by Phe505 p56lck. Several findings indicated that the impact of CD45 in this context was primarily caused by dephosphorylation of downstream signaling targets rather than dephosphorylation of Phe505 p56lck. First, the phosphotyrosine content of Phe505 p56lck was only minimally reduced (less than ~2-fold) in CD45-positive NIH 3T3 cells. Second, CD45 did not cause a global reduction of tyrosine protein phosphorylation in Phe505 Lck-expressing cells. Instead, it seemed to regulate a specific set of tyrosine phosphorylation substrates. These included annexin II, as well as polypeptides of 140 and 120 kDa of yet undetermined identity. In contrast, CD45 had no or little effect on substrates such as cortactin and GAP, as well as the GAP-associated p190 and p62. Third, removal of the Lck unique domain had no consequence on the ability of CD45 to inhibit transformation by Phe505 Lck. Because the unique region was crucial for CD45-mediated dephosphorylation of Lck in other systems (this report and Ref. 23), we feel that this observation provided a most compelling indication that CD45 reduced transformation by Phe505 Lck by acting on downstream signaling events.

These results suggested that CD45 may also have the ability to negatively regulate Lck-mediated signals in T-cells. This notion is consistent with the observation that antibody-mediated co-aggregation of CD45 and the T-cell antigen receptor prevented T-cell receptor-induced intracellular tyrosine protein phosphorylation and T-cell activation (52). Similarly, it supports the finding that some CD45-negative T-cell lines (such as YAC-N1) contained elevated levels of phosphotyrosine-containing proteins prior to T-cell receptor stimulation (29, 53). Hence, CD45 may provide inhibitory signals in T-cells, in addition to its aforementioned positive regulatory role in unstimulated T-cells. Obviously, if these two opposite effects were to occur physiologically, it is expected that they would be differentially regulated at the various stages of T-cell activation.

In summary, we have shown that the ability of CD45 to dephosphorylate the inhibitory tyrosine of p56lck can be reconstituted in a nonlymphoid system, implying that this functional interaction does not require the participation of any other lymphoid-specific component. Unexpectedly, we also found that the catalytic activity of Lck can influence the dephosphorylation of tyrosine 505 by CD45. This observation suggested that physiological changes in the enzymatic activity of p56lck in T-cells may modify its regulation by CD45. Finally, evidence was adduced that CD45 has the capacity to inhibit the impact of Lck activation in vivo, most probably by dephosphorylating downstream signaling targets. In addition to clarifying the basis for the interactions between CD45 and p56lck in T-cells, our experiments permitted us to uncover previously unappreciated mechanisms involved in regulating these interactions.


FOOTNOTES

*   This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Recipient of a Steve Fonyo Studentship from the National Cancer Institute of Canada.
§§   Scientist of the Medical Research Council of Canada. To whom correspondence should be addressed: Rm. 715, McIntyre Medical Sciences Bldg., McGill University, 3655 Drummond St., Montréal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8936; Fax: 514-398-4438; E-mail: veillette{at}medcor.mcgill.ca.
1   The abbreviations used are: SH, Src homology; mAb, monoclonal antibody; GAP, GTPase-activating protein; FACS, fluorescence-activated cell sorter.
2   F. G. Gervais and A. Veillette, unpublished data.

ACKNOWLEDGEMENTS

We thank Pauline Johnson for provision of the expression construct for CD45, Phil Branton for anti-CD45 antibodies, Tony Hunter for the anti-annexin II serum, Tom Parsons for anti-cortactin antibodies, and Tony Pawson for the anti-GAP serum. We also thank the members of our laboratory for critical reading of the manuscript.


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