Reconstitution of interactions between protein-tyrosine phosphatase CD45 and tyrosine-protein kinase p56(lck) in nonlymphoid cells.

To further understand the functional interactions between CD45 and p56(lck) 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 p56(lck) in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56(lck) interfered with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56(lck). The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells was also greatly influenced by the catalytic activity of p56(lck). Indeed, whereas CD45 provoked dephosphorylation of kinase-defective Lck molecules in this system, it failed to stably dephosphorylate kinase-active p56(lck) polypeptides. Finally, our studies showed that CD45 was also able to inhibit the oncogenic potential of a constitutively activated version of p56(lck) in NIH 3T3 cells. This effect did not require the Lck unique domain and apparently resulted from selective dephosphorylation of substrates of activated p56(lck) in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56(lck) in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56(lck)-mediated functions in vivo.

To further understand the functional interactions between CD45 and p56 lck 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 p56 lck in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56 lck interfered with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56 lck . The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells was also greatly influenced by the catalytic activity of p56 lck . Indeed, whereas CD45 provoked dephosphorylation of kinase-defective Lck molecules in this system, it failed to stably dephosphorylate kinase-active p56 lck polypeptides. Finally, our studies showed that CD45 was also able to inhibit the oncogenic potential of a constitutively activated version of p56 lck in NIH 3T3 cells. This effect did not require the Lck unique domain and apparently resulted from selective dephosphorylation of substrates of activated p56 lck in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56 lck in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56 lck -mediated functions in vivo.
p56 lck 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 p56 lck is primarily regulated by phosphorylation of tyrosines 394 and 505 (reviewed in Ref. 5). Phosphorylation at tyrosine 394 activates the catalytic function of p56 lck , by provoking a conformational change in the kinase domain (14). Conversely, phosphorylation at tyrosine 505 inhibits the enzymatic activity of Lck (15)(16)(17), presumably by allowing an intramolecular interaction between the carboxylterminal end and the SH2 domain of the enzyme. Accumulating data indicate that tyrosine 505 phosphorylation is mediated by p50 csk , a tyrosine-protein kinase expressed in all cell types (17)(18)(19)reviewed in Ref. 20). However, tyrosine 505 can also be a site of autophosphorylation. This possibility is suggested by the findings that p56 lck 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 p56 lck (lysine 273 to arginine 273 (Arg 273 ) 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 p56 lck molecules recovered from their CD45-positive counterparts (25)(26)(27)(28)(29). In contrast, expression of CD45 had little or no effect on the tyrosine phosphorylation of two other Src family kinases, p59 fynT and p60 c-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 p56 lck 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 p56 lck or its replacement by the equivalent sequence from another Src family kinase (p59 fynT ) 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 p56 lck . Because the unique domain of p56 lck 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 p56 lck (37)(38)(39)(40).

MATERIALS AND METHODS
Cells-NIH 3T3 mouse fibroblasts and -2 packaging cells were grown in ␣-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 g/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 p56 lck (42) or against the SH3 domain of p56 lck (23) were reported previously. Rabbit antisera directed against annexin II and GTPase-activating protein (GAP) of p21 ras 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). Affinitypurified 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 pH␤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 isothiocyanateconjugated goat anti-rat IgG.
Expression of Lck Polypeptides in NIH 3T3 Cells-cDNAs encoding wild-type p56 lck , lysine 273 to arginine 273 (Arg 273 ) p56 lck , an Arg 273 Lck variant lacking amino acids 16 -62 of the unique domain (⌬16 -62R273 Lck), or tyrosine 505 to phenylalanine 505 (Phe 505 ) Lck were described elsewhere (15,23). An lck cDNA containing the Phe 505 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 -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.
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 (10 3 or 10 4 cells) were seeded in 6-well Costar plates with 10 5 neomycin-resistant NIH 3T3 cells. Foci were counted after 9 days of growth. For growth in soft agar, 2 ϫ 10 4 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.

Generation of CD45-positive Variants of NIH 3T3
Fibroblasts-To further dissect the regulation of p56 lck 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 CD45positive 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 ( CD45 Can Induce Dephosphorylation of p56 lck in NIH 3T3 Cells-Next, CD45-positive and CD45-negative NIH 3T3 cells were infected with retroviruses encoding either wild-type or kinase-defective (Arg 273 ) p56 lck 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 p56 lck 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 p56 lck 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).
In keeping with our previous report (19), wild-type p56 lck (Fig. 2A, lane 2) and Arg 273 p56 lck (lane 3) were tyrosine phosphorylated to similar extents in NIH 3T3 cells lacking CD45. However, although tyrosine phosphorylation of wild-type p56 lck was not decreased by expression of CD45 (lane 4), the phosphotyrosine content of kinase-inactive Lck molecules was reduced ϳ5-fold (lane 5). The differential impact of CD45 on wild-type and Arg 273 p56 lck 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 32 P i , 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 p56 lck by cyanogen bromide was complete and that equivalent amounts of Lck peptides were applied in each lane.
Cleavage of p56 lck 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)(46)(47). In CD45-negative fibroblasts, both wild-type (lane 1) and kinase-defective (lane 2) p56 lck 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 carboxylterminal phosphorylation of Arg 273 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 kinaseinactive 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 (ϳ8-fold) increase in the phosphotyrosine content of Arg 273 Lck in CD45positive NIH 3T3 cells (Fig. 3A, compare lanes 2 and 4). Cyanogen bromide cleavage analysis (Fig. 3B) (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 tyrosineprotein kinase can transphosphorylate tyrosine 394 in vivo (19,48,49).
The Unique Domain of p56 lck 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 p56 lck was also necessary for the effect of CD45 in fibroblasts. Arg 273 Lck polypeptides lacking residues 16 -62 (⌬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 ⌬16 -62R273 Lck (lane 3) was tyrosine phosphorylated to nearly the same extent as Arg 273 p56 lck (lane 1) in CD45-negative cells. In cells expressing CD45, however, the tyrosine phosphorylation of ⌬16 -62R273 Lck (lane 4) was only reduced ϳ1.5-fold, in comparison with the ϳ5-fold decrease noted above for Arg 273 Lck (lane 2; Fig. 2A, lanes 3 and 5). Complementary peptide mapping analyses confirmed that, unlike Arg 273 Lck (Fig. 4B, lane 2), ⌬16 -62R273 Lck remained prominently phosphorylated at tyrosine 505 in CD45-positive cells (lane 4).
Because CD45 is a membrane-bound phosphatase, we wished to ensure that the association of p56 lck 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 Arg 273 p56 lck (Fig 4C, lanes 1 and 3), more than 80% of ⌬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 p56 lck in NIH 3T3 Cells-Introduction of a constitutively activated version of p56 lck (tyrosine 505 to phenylalanine 505 p56 lck ) in NIH 3T3 cells causes marked tyrosine phospho- rylation 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 p56 lck molecules, CD45-positive NIH 3T3 cells were infected with retroviruses encoding Phe 505 p56 lck . CD45-negative neomycin-resistant NIH 3T3 cells (Neo) were used as control recipient. Immunoblot analyses showed that all infected cells expressed comparable amounts of Phe 505 p56 lck (data not shown; see 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 Phe 505 p56 lck without CD45 (Fig. 5C) were rounded and refractile and displayed multiple neuronal-like processes (15,16). By contrast, cells expressing Phe 505 p56 lck 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 Phe 505 Lck alone, cells expressing Phe 505 Lck and CD45 could form foci in monolayers. However, the foci were ϳfive times smaller than those generated by Phe 505 Lck-expressing cells. Furthermore, although fibroblasts expressing Phe 505 p56 lck formed large colonies in soft agar, cells expressing Phe 505 Lck and CD45 failed to grow under this condition.
To elucidate the mechanism by which CD45 prevented oncogenic transformation by Phe 505 p56 lck , we studied its impact on the accumulation of phosphotyrosine-containing proteins (Fig.  6). Anti-phosphotyrosine immunoblotting of total cell lysates demonstrated that cells expressing Phe 505 Lck and CD45 (Fig.  6A, lane 4) contained lower amounts of phosphotyrosine-containing proteins than cells expressing Phe 505 p56 lck 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 phospho-tyrosine 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 Phe 505 Lck and CD45 (Fig. 6B, lane 4) in contrast to cells containing Phe 505 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 Phe 505 p56 lck -expressing cells (Fig. 6A, lane 4).
We wanted to determine whether the effect of CD45 on Phe 505 Lck-expressing cells was due to dephosphorylation of Phe 505 p56 lck or to dephosphorylation of downstream targets. To this end, the phosphotyrosine content of Phe 505 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 Phe 505 p56 lck by ϳ2-fold. To ascertain whether these changes were due to dephosphorylation of tyrosine 394, the positive regulatory site of p56 lck , peptide mapping studies were conducted (Fig. 7B). These analyses failed to show any reduction in phosphorylation of the C2 fragment of p56 lck , which contains tyrosine 394. Although a small decrease in C1 phos-phorylation 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 Phe 505 p56 lck but rather by dephosphorylating its substrates. To help support this idea, the impact of CD45 on transformation by a variant of Phe 505 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 p56 lck (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 ⌬16 -62F505 Lck. Cells containing ⌬16 -62F505 Lck without CD45 were morphologically transformed in a manner analogous to Phe 505 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 Phe 505 Lck-expressing cells (Table II). Anti-phosphotyrosine immunoblotting of total cell lysates showed that ⌬16 -62F505 Lck-expressing cells also possessed elevated levels of phosphotyrosine-containing proteins (Fig. 8, lane 5).
As was the case for cells containing Phe 505 Lck and CD45, cells expressing ⌬16 -62F505 Lck and CD45 exhibited a morphology that was intermediate between those of Neo cells and ⌬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 ⌬16 -62F505 Lck-expressing cells (Fig. 8, lane 6) in manner similar to that observed in cells expressing Phe 505 p56 lck (lane 4). Hence, these data indicated that CD45 inhibited Phe 505 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 p56 lck in a nonlymphoid system. The results of our experiments showed that expression of the R0 isoform of CD45 in NIH 3T3 fibroblasts caused a ϳ5-fold decrease in the extent of tyrosine 505 phosphorylation of kinase-defective (Arg 273 ) p56 lck 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 p56 lck 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 p56 lck 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 p56 lck . Specifically, they also demonstrated that CD45-associated protein, a protein that can associate with both CD45 and p56 lck (37)(38)(39)(40)(41) and is not expressed in NIH 3T3 cells, 2 is not critical for the action of CD45 on p56 lck . 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, Arg 273 , or Phe 505 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 p56 lck can influence the ability of CD45 to stably dephosphorylate tyrosine 505. It is conceivable that the enzymatic function of p56 lck antagonizes the effect of CD45 by allowing autophosphorylation at tyrosine 505 (21,22). Alternatively, the enzymatic activity of Lck may facilitate the recruitment of p50 csk , the tyrosine-protein kinase normally responsible for phosphorylating tyrosine 505 (Refs. [17][18][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 p56 lck 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 p56 lck 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 p56 lck (Phe 505 Lck) was also evaluated. Contrary to cells containing Phe 505 p56 lck alone, cells harboring Phe 505 p56 lck 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 Phe 505 p56 lck . Several findings indicated that the impact of CD45 in this context was primarily caused by dephosphorylation of downstream signaling targets rather than dephosphorylation of Phe 505 p56 lck . First, the phosphotyrosine content of Phe 505 p56 lck was only minimally reduced (less than ϳ2-fold) in CD45-positive NIH 3T3 cells. Second, CD45 did not cause a global reduction of tyrosine protein phosphorylation in Phe 505 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 GAPassociated p190 and p62. Third, removal of the Lck unique domain had no consequence on the ability of CD45 to inhibit transformation by Phe 505 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 Phe 505 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 p56 lck 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 p56 lck 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 p56 lck in T-cells, our experiments permitted us to uncover previously unappreciated mechanisms involved in regulating these interactions.