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The Journal of Immunology, 2007, 178: 2056-2064.
Copyright © 2007 by The American Association of Immunologists, Inc.

Changes in the Role of the CD45 Protein Tyrosine Phosphatase in Regulating Lck Tyrosine Phosphorylation during Thymic Development1

Rustom Falahati2,* and David Leitenberg3,*,{dagger}

* Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, DC 20037; and {dagger} Children’s National Medical Center, Washington, DC 20010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD45-dependent dephosphorylation of the negative regulatory C-terminal tyrosine of the Src family kinase Lck, promotes efficient TCR signal transduction. However, despite the role of CD45 in positively regulating Lck activity, the distinct phenotypes of CD45 and Lck/Fyn-deficient mice suggest that the role of CD45 in promoting Lck activity may be differentially regulated during thymocyte development. In this study, we have found that the C-terminal tyrosine of Lck (Y505) is markedly hyperphosphorylated in total thymocytes from CD45-deficient mice compared with control animals. In contrast, regulation of the Lck Y505 phosphorylation in purified, double-negative thymocytes is relatively unaffected in CD45-deficient cells. These changes in the role of CD45 in regulating Lck phosphorylation during thymocyte development correlate with changes in coreceptor expression and the presence of coreceptor-associated Lck. Biochemical analysis of coreceptor-associated and nonassociated Lck in thymocytes, and in cell lines varying in CD4 and CD45 expression, indicate that CD45-dependent regulation of Lck Y505 phosphorylation is most evident within the fraction of Lck that is coreceptor associated. In contrast, Lck Y505 phosphorylation that is not coreceptor associated is less affected by the absence of CD45. These data define distinct pools of Lck that are differentially regulated by CD45 during T cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD45 protein tyrosine phosphatase is an important regulator of T lymphocyte development and activation. However, the precise role(s) of CD45 in regulating the different aspects of T cell activation, and the mechanisms that regulate CD45 activity remain poorly understood. Experiments using CD45-deficient cell lines and animals indicate that CD45 ultimately has a positive effect on T cell activation (1). In CD45-deficient mice and humans, there is a profound block in thymic development demonstrating the importance of CD45 in promoting T cell activation and development (2, 3, 4, 5). The defects in T cell activation seen in the absence of CD45 are thought to be secondary to decreased activity of the Src family kinase Lck (and perhaps Fyn). Lck is the best defined substrate for CD45, and is positively regulated by CD45-dependent dephosphorylation of a negative regulatory C-terminal tyrosine residue (Y505) maintaining an active or "open" conformation (6). This site is hyperphosphorylated in CD45-deficient T cell lines and in CD45-deficient thymocytes (7, 8, 9, 10, 11), and reconstitution with constitutively "active" Lck in which the C-terminal tyrosine is mutated to phenylalanine, is able to restore initial TCR signal transduction events and T cell development in the absence of CD45 (12, 13, 14). Acting in opposition to CD45, is the C-terminal Src kinase (Csk)4 that phosphorylates the C-terminal tyrosine of Lck and negatively regulates T cell activation (15, 16). Thus, the roles of CD45 and Csk in regulating Lck activity are in dynamic equilibrium. Regulatory mechanisms that control CD45 and Csk activity and/or access to Lck may have important effects on the sensitivity of TCR recognition, and outcomes of T cell activation.

Despite the recognized role of CD45 in regulating SFK activity, there are several unresolved issues regarding the requirement for CD45 in promoting Lck activity in vivo. For example, thymocyte development is differentially affected in CD45 and Lck/Fyn-deficient mice. In the absence of CD45, there is a severe block during positive selection and in the development of mature single-positive T cells, whereas pre-TCR signaling and the development of double-positive (DP) T cells is less severely affected (2, 3, 4). This contrasts with studies in Lck or Lck/Fyn-deficient mice, where there is a more severe defect in pre-TCR signaling resulting in a failure to transition from the double-negative (DN) to DP stage of thymic development (17, 18, 19). The different phenotypes of Lck and CD45-deficient mice suggest that the role of CD45 in regulating Lck activation may be developmentally regulated during thymocyte development. In this study, we have found that regulation of C-terminal tyrosine phosphorylation of coreceptor-associated Lck in DP cells is CD45-dependent. In contrast, regulation of Lck Y505 phosphorylation in DN thymocytes, as well as nonraft, non-coreceptor-associated Lck, is relatively independent of CD45 expression. In total, these data define distinct pools of Lck that are differentially regulated by CD45 and coreceptors during thymic development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CD45 exon 9 null mice were purchased from the The Jackson Laboratory and have been backcrossed 7–9 times onto the B10.Br background (3). All mice were bred and maintained at the George Washington University animal facility (Washington, DC). All animal procedures conform to institutional animal protocol guidelines.

Cell lines

Description of the BW5147 AKR thymoma lines with or without CD45 and/or CD4 have been described previously (20). In this study, we have isolated and maintained a CD4–CD45RO+ cell line derived from the CD4+CD45RO+ cells by negative selection using magnetic beads coated with anti-CD4. Identity and purity of each cell line was evaluated during each experiment, and expression of CD45RO and CD4 were maintained at similar levels in the different cell lines as appropriate.

Preparation of DN thymocytes

DN thymocytes were purified from total thymocytes from CD45+/– and CD45–/– mice using immunomagnetic negative selection with Abs against CD4 (GK1.5) and CD8{alpha} (53-6.72) followed by incubation with anti-mouse and anti-rat Ig-coated magnetic beads (Perspective Diagnostics). Purity of the recovered CD4–CD8–TCRbetalow DN thymocytes was >90% as determined by flow cytometric analysis.

Cell lysis, immunoprecipitation, and immunoblotting

Cell lysates containing an equal number of cells were prepared by lysis in TNE buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4), supplemented with complete Mini protease inhibitors (Roche Applied Science), 1 mM sodium orthovanadate, and either 1% Nonidet P-40 (NP-40) or 1% N-dodecyl-beta-D-maltoside (Maltoside) as indicated. Postnuclear, detergent soluble, extracts were immunoprecipitated with anti-CD4 and/or anti-CD8, and cleared cell lysates from these immunoprecipitates with same cell equivalence to total cell lysates were also saved. Western blot analysis was done following SDS polyacrylamide electrophoresis and transferred onto nitrocellulose paper (Bio-Rad). The phosphorylation status of the regulatory tyrosines (Y505 and Y394) of Lck were detected by phosphospecific Abs (Cell Signaling Technology). Phosphorylation of TCR{zeta} was detected with a pan phosphotyrosine Ab (4G10; Upstate Biotechnology). Total Lck, TCR{zeta}, and Csk were detected using rabbit polyclonal Abs (21) (Cell Signaling Technology). Immunoblots were developed using goat anti-rabbit or anti-mouse Ig-coupled HRP and visualized with the ECL chemiluminescent detection system. Bands from immunoblots were quantified by densitometry (Molecular Dynamics), and the relative degree of Lck phosphorylation levels were corrected for total amounts of Lck.

Lipid raft separation

Lipid rafts were isolated following lysis in TNE buffer containing 1% Brij 58 and separation on a sucrose density gradient as described previously (22), with slight modifications. Lysates were mixed with an equal volume of 80% sucrose in TNE buffer, and then sequentially overlaid with 35 and 5% sucrose in TNE buffer. Individual fractions were harvested following centrifugation at 100,000 x g at 4°C for 3–4 h, and then pooled into raft (fractions 2–4) and nonraft (fractions 10–12) fractions. Raft and nonraft fractions were solubilized in 1% N-dodecyl-beta-D-maltoside and (in some experiments) concentrated using the PAGEprep Advance Kit (Pierce). The fractions were evaluated by SDS-PAGE as described above.

Pervanadate treatment

A concentrated solution of 1 mM pervanadate was freshly prepared by addition of sodium orthovanadate (Calbiochem) and hydrogen peroxide (Sigma-Aldrich) as described previously (23). Intact cells were treated with 100 µM pervanadate for 1 min at 37°C, and cell lysates were immediately prepared as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD45-dependent regulation of Lck tyrosine phosphorylation

Previous phosphopeptide mapping studies in CD45-deficient tumor cell lines have indicated that Lck is hyperphosphorylated at the negative regulatory C-terminal tyrosine residue, Y505, as well as the positive regulatory site within the catalytic domain of Lck (Y394) (11, 24). These data suggest that CD45 may have both positive and negative regulatory effects on Lck activity depending on the cell line examined and perhaps the activation state of the cell. Indeed, some in vitro studies of Lck kinase activity in CD45-deficient cell lines have found little or no positive role for CD45 on overall Lck kinase activity (11). In vitro kinase assays of Lck activity from primary thymocytes from CD45-deficient mice have also found that total Lck kinase activity is paradoxically increased in CD45-deficient cells compared with wild-type controls (25), despite the profound block in thymocyte development seen in the absence of CD45. The reason for this apparent paradoxical effect of CD45 on Lck kinase activity is unclear and suggests that in vitro kinase assays may not always reflect in vivo activity. In addition, analysis of CD45-dependent regulation of Lck activity may be complicated by the fact that there may be distinct pools of Lck within a cell that are differentially regulated by CD45 and have distinct enzymatic activities. To directly assess the role of CD45 in regulating Lck tyrosine phosphorylation in primary cells, we have used Abs that specifically detect tyrosine phosphorylation at either the negative regulatory C-terminal site (Y505) or the positive regulatory within the catalytic domain of Lck (Y394) in primary thymocytes from CD45-deficient animals. As shown in Fig. 1A, Lck Y505 is markedly hyperphosphorylated in CD45-deficient cells compared with wild-type control cells, whereas there was little if any effect of CD45 deficiency on Lck Y394 phosphorylation. These data indicate that, in primary thymocytes, CD45 predominantly regulates the phosphorylation of the negative regulatory C-terminal residue of Lck. To assess whether hyperphosphorylation of Lck Y505 is associated with decreased activity in vivo, we also examined phosphorylation of endogenous TCR-associated {zeta}-chain in wild-type and CD45-deficient thymocytes. As seen in a previous study and consistent with decreased in vivo Lck activity, there is a marked decrease in basal TCR{zeta}-chain (p21) phosphorylation in CD45-deficient animals compared with CD45+/– control thymocytes (Fig. 1B) (26).


Figure 1
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  		FIGURE 1. Regulation of Lck Y505 phosphorylation and basal TCR{zeta}-chain phosphorylation in CD45-deficient thymocytes. A, NP-40 soluble cell lysates (1%) from 2 x 106 total thymocytes from CD45+/– or CD45–/–, mice were analyzed for pY505 Lck by Western blot. The pY505 blot was then stripped and probed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification and is indicated below each sample lane. B, Cell lysates from 2 x 106 total thymocytes from CD45+/– or CD45–/– mice were analyzed for CD3{zeta}-chain (p21) phosphorylation using anti-phosphotyrosine Ab, then stripped and probed for total CD3{zeta} (p16, nonphosphorylated) by Western blot. The ratio of pCD3{zeta} (p21) to total CD3{zeta} was assessed by densitometric quantification. These data are representative of >3 independent experiments.

 
Differential role of CD45 in regulating C-terminal tyrosine phosphorylation of Lck (Y505) by CD45 in purified DN thymocytes compared with total thymocytes

Although it is clear from the literature and the above data that CD45 can positively regulate Lck activity, comparison of thymocyte development in Lck-deficient and CD45-deficient animals suggest that the role of CD45 in regulating Lck function is developmentally regulated. Previous studies have found that thymocyte development is blocked at the DP stage in CD45-deficient mice, whereas the transition from DN T cells to DP cells is less severely impacted (2, 3, 4). These data suggest that pre-TCR signaling can occur in the absence of CD45. In contrast, studies in mice deficient in Lck or Lck/Fyn, the major substrates of CD45, have a severe block at the DN stage of thymocyte development (17, 18, 19). In total, these data suggest that the role of CD45 in promoting Lck activity is more stringent at the DP stage and is required for positive selection, whereas CD45 is partially dispensable for Lck activity at the DN stage of thymocyte development. To directly assess the role of CD45 in regulating C-terminal tyrosine (Y505) phosphorylation of Lck at different stages of thymocyte development, we compared Lck Y505 phosphorylation within total thymocytes to purified DN thymocytes from CD45+/– and CD45–/– mice. As shown in Figs. 1 and 2A, in total thymocytes (consisting of ~85% DP thymocytes), Lck Y505 was hyperphosphorylated in the absence of CD45. This was in marked contrast to purified DN thymocytes, where Lck Y505 phosphorylation was similar in cells from both CD45+/– and CD45–/– mice (Fig. 2A). DN thymocyte subsets from CD45-intact and CD45-deficient mice were similar as shown by the CD44 and CD25 expression pattern, although there is a modest increase in the percentage of cells at the DN3 stage of thymic development as described previously (3, 4) (Fig. 2B). These experiments were typically done comparing thymocytes from CD45–/– and CD45+/– littermates. Because CD45+/– cells have ~40% less CD45 surface expression when compared with CD45+/+ cells, we repeated the analysis comparing CD45+/+ and CD45–/– thymocytes. A decreased role for CD45 in regulating Lck Y505 phosphorylation in DN cells was also evident upon comparison of CD45+/+ and CD45–/– thymocytes (data not shown).


Figure 2
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  		FIGURE 2. Differential role of CD45 in regulating Lck Y505 phosphorylation in total thymocytes compared with DN thymocytes. A, Total thymocytes (2 x 106) and DN thymocytes from CD45+/– or CD45–/– mice were analyzed for pY505 Lck by Western blot as in Fig. 1. The pY505 blot was then stripped and probed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification. The data shown are representative of >3 independent experiments. B, Representative flow cytometry data assessing CD4 and CD8 expression of the total thymocyte population from CD45+/– or CD45–/– mice (left). Flow cytometric analysis of CD44 and CD25 expression of purified DN thymocytes from CD45+/– and CD45–/– mice (right). C, Total thymocytes (1 x 106) and DN thymocytes from CD45+/+ or CD45–/– mice were analyzed for Csk expression by Western blot. The blot was then stripped and reprobed for total Lck to control for changes in T cell loading.

 
Analysis of Lck Y505 phosphorylation in both CD45-deficient and control DN thymocytes indicates that there is only a small increase in basal tyrosine phosphorylation of Lck Y505 compared with total thymocytes from CD45+ animals (lane 1 vs lanes 3 or 4, Figure 2A). Thus, in addition to illustrating a differential requirement for CD45 in regulating Lck phosphorylation during development, these data suggest that in the absence of CD45, there is a reduction in the tyrosine phosphorylation of Lck 505 in the DN cell population compared with DP cells. This may reflect a difference in access to a tyrosine kinase (such as Csk) at the DN stage of development. However, evaluation of Csk levels in total and DN thymocytes by Western blot demonstrate a modest increase in Csk expression in DN cells, suggesting that Csk has the potential to act on Lck early in thymocyte development (Fig. 2C). This is consistent with previous studies in animals with a targeted deletion of Csk in developing thymocytes that found an active role for Csk in regulating beta-selection and development of DP thymocytes (15, 27).

CD45 preferentially regulates phosphorylation of coreceptor-associated Lck compared with non-coreceptor-associated Lck

An obvious distinction between DN and DP thymocytes is coreceptor expression. CD4 and CD8 coreceptor both associate with Lck and may alter access of Lck to both CD45 and Csk, the main regulators of Lck-Y505 phosphorylation. Indeed, previous reports have suggested that CD45 is closely associated with CD4, which may promote CD45 access to CD4-associated Lck (20, 28, 29). Alternatively, the different roles of CD45 in regulating Lck tyrosine phosphorylation during thymocyte maturation may be an intrinsic characteristic of cells at different stages of thymocyte development, and may be regulated independently of coreceptor expression.

To address the role of CD4 coreceptor in modifying CD45-dependent regulation of Lck phosphorylation, we evaluated the phosphorylation of Lck Y505 in individual CD45-deficient thymoma cell lines (BW cells) transfected with or without CD45 and CD4. As shown in Fig. 3A, CD4 expression modulated the Lck hyperphosphorylation seen in the absence of CD45. In the presence of CD45, Lck was basally hypophosphorylated in both CD4+ and CD4– cell lines. However, in CD45-deficient cell lines, the CD4+ cells exhibited increased Lck Y505 phosphorylation compared with cells that were CD4–. These data suggest that CD4 expression promotes CD45-dependent regulation of Lck phosphorylation.


Figure 3
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  		FIGURE 3. CD45 preferentially regulates phosphorylation of CD4 coreceptor-associated Lck. A, A total of 5 x 106 cells from the indicated BW cell lines were lysed in 1% NP-40 lysis buffer, and analyzed for pY505 Lck by Western blot and then stripped and reprobed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification and normalized for the total amount of Lck. B, CD4-cleared cell lysates from 5 x 106 cell equivalents or CD4 immunoprecipitates (CD4 IP) from 12.5 x 106 cells were separated by SDS-PAGE and blotted for pY505 Lck and total Lck as in A. The Western blot data shown is from the same blot and exposure so that total amounts of Lck are comparable. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification and normalized for the total amount of Lck. The data shown are representative of three independent experiments. C, NP-40 soluble cell lysates from CD45–/– or CD45+/– total thymocytes was immunoprecipitated with Abs to CD4 and CD8, or control Ab to CD44. Following immunoprecipitation, the cleared cell lysates were analyzed for Lck pY505 by Western blot, and then stripped and reprobed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification. As a control to insure proper clearing of coreceptor, the presence of CD4 in each cleared cell lysate was determined by Western blot. The data shown are representative of two independent experiments.

 
Similar results are found when CD45-dependent Lck Y505 phosphorylation of CD4-associated Lck is directly compared with non-coreceptor-associated Lck obtained from cell lysates precleared with Ab to CD4 (Fig. 3, B and C). As seen in the total cell lysates from the CD45–CD4– cell line presented in Fig. 3A, there is a relatively modest increase in non-coreceptor-associated Lck Y505 phosphorylation in the CD4-cleared cell lysates from CD45-deficient cells in both CD4+ and CD4– cell lines (Fig. 3B). However, phosphorylation of Lck Y505 that is CD4-associated is significantly more dependent on CD45 expression, when compared with non-CD4-associated Lck (Fig. 3B, lanes 3 vs 7).

To directly compare CD45-dependent regulation of coreceptor-associated Lck vs non-coreceptor-associated Lck tyrosine phosphorylation in primary cells, coreceptor (CD4 and CD8) preclearing experiments in CD45+/– and CD45–/– thymocytes were performed. As seen in the cell line experiments, we observed that Lck Y505 phosphorylation from cleared cell lysates, consisting of non-coreceptor-associated Lck, was less affected by loss of CD45 expression compared with lysates cleared with a control Ab (Fig. 3C). In contrast, coreceptor-associated Lck was profoundly hyperphosphorylated at Y505 in the absence of CD45 expression (data not shown). As a control for preclearing, cleared cell lysates were free of coreceptor when blotted and probed for the presence of CD4 (Fig. 3C).

In total, these data suggest that the phosphorylation status of CD4-associated Lck is regulated by CD45, whereas the phosphorylation of Lck that is not associated with CD4 is less affected by CD45 deficiency. In addition, the data suggest that in the absence of CD45, CD4-associated Lck Y505 is phosphorylated more readily than non-CD4-associated Lck. Although Csk expression was expressed at identical levels in all BW cell lines (data not shown), the majority of cellular Csk is located in the cytoplasm. It is possible that CD4 may promote membrane association of Lck and subsequent access to the fraction of Csk, which is membrane associated. This may be especially true for raft-associated Lck, because one mechanism by which Csk is targeted to the plasma membrane is through association with the lipid raft-associated molecule, cbp/PAG (16).

Differential regulation of Lck Y505 phosphorylation in lipid raft membrane microdomains and nonraft compartments

As noted above, Lck can also be compartmentalized within lipid raft membrane microdomains in addition to association with CD4. Initial reports in tumor cell lines suggested that raft-resident Lck was constitutively hyperphosphorylated due to the exclusion of CD45 from rafts and presumably due to raft targeting of Csk tyrosine kinase by PAG/cbp (30). However, more recent reports have suggested that a small percentage of CD45 has access to lipid raft domains and may regulate raft-resident Lck tyrosine phosphorylation (31, 32). To clarify this issue in primary cells, we purified lipid raft and nonraft cell fractions from CD45+/– and CD45–/– thymocytes by sucrose density gradient centrifugation. As shown in Fig. 4A, CD45 regulates both raft and nonraft-associated Lck Y505 phosphorylation in cell lysates from total thymocytes. However, the majority of Lck protein from total thymocytes is in the nonraft fraction.


Figure 4
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  		FIGURE 4. CD45-dependent regulation of Lck Y505 phosphorylation within lipid raft and nonraft compartments. A, Lipid raft and nonlipid raft fractions were isolated from 100 x 106 CD45+/– or CD45–/– thymocytes following lysis in 1% Brij 58 and separation on a sucrose density gradient. Pooled raft and nonraft fractions were solubilized with 1% N-dodecyl-beta-D-maltoside and evaluated for Lck pY505 by Western blot. Quantification of the relative change in Lck pY505 between CD45+/– and CD45–/– lysates is presented following independent normalization to the total Lck in raft and nonraft fractions of the CD45+/– cell lysates. B, Lipid raft and nonlipid raft-pooled fractions from 100 x 106 of the indicated BW cell lines were analyzed following lysis in 1% Brij 58 and separation on a sucrose density gradient as in A. Pooled raft and nonraft fractions were evaluated for Lck pY505 by Western blot, and then stripped and probed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification.

 
Because differential expression of CD4 may alter the relative proportions of Lck within lipid raft membrane domains and affect access to CD45 and Csk, we also assessed the role of CD4 in regulating CD45-dependent dephosphorylation of Lck-Y505 within lipid raft and nonraft fractions using the BW cell lines described in Fig. 3. As shown in Fig. 4B and similarly to the total thymocyte data (Fig. 4A), we observed that CD45 regulates Lck Y505 phosphorylation in both lipid raft and nonlipid raft compartments. Interestingly, we found that the expression of CD4 profoundly affects the distribution of Lck protein in the lipid raft compartment. In CD4-negative cells, there was a significant increase in the amount of total Lck in raft membrane fractions, and corresponding loss in nonraft fractions, compared with CD4+ cells. However, when normalized for total Lck protein, raft resident Lck Y505 phosphorylation was regulated by CD45 similarly to Lck from CD4+ cells (Fig. 4B, left panels). This result was in contrast to the Lck found in nonraft fractions, where CD4 expression promotes regulation of Lck Y505 phosphorylation by CD45 (Fig. 4B, right panels, lanes 3 vs 4). These data suggest that the role of CD4 in modifying CD45-dependent regulation of Lck Y505 phosphorylation in the BW cells is particularly important in nonraft microdomains. This is consistent with the data in Fig. 3, in which CD4 regulation of Lck Y505 phosphorylation was examined in NP-40 soluble lysates in which raft components are underrepresented (see below). Because membrane-associated Csk can be localized within lipid raft membrane microdomains by associating with PAG/cbp, we assessed changes in raft-associated Csk in cell lines varying in CD4 and CD45. Csk expression and raft association in the BW cells and was not affected by changes in CD45 or CD4 expression (data not shown).

Of note in this Western blot of the raft fractions from the BW cells, is an additional protein that we have identified as the Src family kinase Fyn (Fig. 4B). In contrast to Lck, phosphorylation of the C-terminal tyrosine of Fyn is not significantly increased in the CD45-deficient BW cells.

Regulation of Lck Y505 phosphorylation in primary DN thymocytes in raft and nonraft cellular compartments

Because differences in the regulation of signal transduction between pre-TCR and mature TCR signaling complexes have been postulated to be partially due to differences in lipid raft association (33), we also evaluated the role of lipid rafts in modulating CD45-dependent regulation of Lck phosphorylation in DN thymocytes. In these experiments, we indirectly assessed the role of CD45 in regulating raft-resident Lck in DN thymocytes using the enhanced ability of N-dodecyl-beta-D-maltoside to solubilize lipid raft like membrane compartments compared with NP-40 (34). This approach was necessary due to the difficulty in obtaining sufficient numbers of purified DN thymocytes to separate lipid raft and nonraft Lck pools by sucrose density gradient. When DN thymocytes are lysed in 1% N-dodecyl-beta-D-maltoside containing lysis buffer, there is an increase in the amount of total Lck protein recovered compared with cell lysates prepared using 1% NP-40, consistent with it’s enhanced ability to solubilize detergent insoluble membrane fractions (Fig. 5). Increases in protein recovery are seen with other raft resident proteins such as LAT and PAG, but not with proteins that are largely nonraft associated such as TCR{zeta} or ERK (data not shown). The increase in Lck protein upon solubilization of the DN thymocytes in 1% N-dodecyl-beta-D-maltoside compared with 1% NP-40 is also consistent with the data in BW cells that CD4 coreceptor expression promotes Lck compartmentalization in nonraft domains (Fig. 4B). Indeed, in contrast to DN thymocytes, solubilization of total thymocytes in 1% N-dodecyl-beta-D-maltoside did not significantly change Lck protein recovery or tyrosine phosphorylation status compared with NP-40 lysates (Fig. 5), consistent with the data indicating that the majority of Lck in total thymocytes is nonraft associated (Fig. 4A).


Figure 5
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  		FIGURE 5. Role of CD45 in regulating lipid raft and nonraft Lck Y505 phosphorylation in DN thymocytes using differential detergent solubilization. Total or purified DN thymocytes (1 x 106) from CD45+/– or CD45–/– mice were analyzed for pY505 Lck by Western blot following lysis in either 1% NP-40 or 1% N-dodecyl-beta-D-maltoside (MALT). The pY505 blot was then stripped and probed for total Lck. The ratio of Lck pY505 to total Lck was assessed by densitometric quantification and normalized to CD45+/– cells for both total and DN cells. These data are representative of three independent experiments.

 
When Lck Y505 phosphorylation from purified DN CD45–/– and CD45+/– thymocytes is compared with 1% N-dodecyl-beta-D-maltoside soluble lysates, there is little overall change in the role of CD45 in regulating Lck phosphorylation when compared with 1% NP-40 soluble lysates (Fig. 5) (see also Fig. 6B). Regardless of the detergent used for cell lysis, the role of CD45 in regulating Lck Y505 phosphorylation in DN thymocytes is considerably less than that seen in cell lysates from CD45-deficient total thymocytes (Figs. 1, 2, 4A, and 5). These data differ somewhat from that obtained from the BW cells. In the BW cell model, CD45 regulates CD4-associated Lck phosphorylation in nonraft domains, whereas raft-associated Lck Y505 phosphorylation is also regulated by CD45, but is independent of CD4 expression. In DN thymocytes, lysis in maltoside causes an increase in Lck protein detectable by Western blot, consistent with an increase of total Lck protein in lipid rafts in DN cells. However, DN thymocytes from CD45-deficient mice continue to exhibit relatively little difference in Lck Y505 phosphorylation when compared with CD45+ DN cells.


Figure 6
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  		FIGURE 6. Differential regulation of Lck Y505 phosphorylation in response to pervanadate treatment in DN thymocytes. A, Total thymocytes (2 x 106) from CD45+/+ mice were stimulated with 100 µM pervanadate for 1 min or left untreated and then analyzed for pY505 Lck or total phosphotyrosine by Western blot following lysis in 1% N-dodecyl-beta-D-maltoside. The pY505 blot was then stripped and probed for total Lck, and the ratio of Lck pY505 to total Lck was assessed by densitometric quantification. B, Total (1 x 106) purified DN thymocytes from CD45+/– or CD45–/– mice were stimulated with 100 µM pervanadate for 1 min or left untreated and then analyzed for pY505 Lck by Western blot following lysis in 1% N-dodecyl-beta-D-maltoside. The pY505 blot was then stripped and probed for total Lck, and the ratio of Lck pY505 to total Lck was assessed by densitometric quantification. The same blot was also assessed for the induction of total protein tyrosine phosphorylation by Western blot (bottom panel). These data are representative of three independent experiments. C, Total thymocytes from CD45+/+ mice were stimulated with pervanadate as shown in B. Maltoside soluble lysates were then immunoprecipitated with Ab to CD4 and CD8 and coreceptor-associated Lck, or Lck from CD4/CD8-cleared cell lysates were then analyzed for changes in Lck Y505 phosphorylation by Western blot. The ratio of Lck Y505 phosphorylation to total Lck was assessed by densitometric quantification.

 
Differential effect of pervanadate treatment on Lck Y505 phosphorylation during thymic development

The relatively small role of CD45 in regulating Lck tyrosine phosphorylation in DN thymocytes compared with DP thymocytes mirrors the effect of CD45 deficiency on thymic development, where there is a severe block in development at the DP stage and a relatively modest block at the DN stage (2, 3, 4). The differential requirement for CD45 in regulating Lck Y505 phosphorylation in these cell populations corresponds with the presence of coreceptor-associated Lck (Fig. 3). Phosphorylation of coreceptor-associated Lck is affected by changes in CD45 expression, whereas in Lck in cells that lack coreceptor expression or in the non-coreceptor-associated pool of Lck, the regulation of Lck Y505 tyrosine phosphorylation is less dependent on CD45. These data suggest that an additional protein tyrosine phosphatase may regulate non-coreceptor-associated Lck tyrosine phosphorylation at the DN stage of thymic development. Alternatively, although Csk is expressed in DN cells, it is possible that Csk (or another unknown kinase) is less active early in thymic development and/or does not have efficient access to the non-coreceptor-associated pool of Lck, thus decreasing the requirement for CD45 to maintain Lck in a primed active configuration.

To investigate these possibilities, we initially treated total thymocytes with the protein tyrosine phosphatase inhibitor, pervanadate. Consistent with previous reports, pervanadate treatment inhibits protein tyrosine phosphatase activity, and induces tyrosine phosphorylation of Lck Y505 and other signaling intermediates in T lymphocytes (Fig. 6A) (35). If an alternative protein tyrosine phosphatase is involved in regulating Lck Y505 phosphorylation in DN thymocytes, pervanadate treatment would be expected to promote hyperphosphorylation of Lck in CD45-deficient cells. Alternatively, if maintenance of steady-state levels of Lck tyrosine phosphorylation in DN cells is independent of protein tyrosine phosphatase activity, pervanadate treatment should have little or no effect. As seen in Fig. 6B, pervanadate treatment did not induce hyperphosphorylation of Lck from purified DN thymocytes from either CD45-deficient or CD45+/– mice. Analysis of pervanadate-dependent increases in total tyrosine phosphorylation in the DN cell populations indicate that pervanadate treatment was sufficient to block endogenous protein tyrosine phosphatase activity similarly to that seen in total thymocytes.

Conceptually, similar results are obtained when the phosphorylation of Lck Y505 from coreceptor-cleared thymocyte lysates is compared with Lck associated with coreceptor following phosphatase inhibition by pervanadate treatment (Fig. 6C). In this experiment, pervanadate promoted hyperphosphorylation of Lck Y505 in Lck coimmunoprecipitated with coreceptor, whereas Lck from coreceptor-cleared lysates was significantly less affected by pervanadate treatment. These data suggest that phosphorylation of coreceptor and non-coreceptor-associated Lck are differentially affected by inhibition of phosphatase activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In summary, the above data indicate that the role of CD45 in controlling Lck Y505 phosphorylation is developmentally regulated during thymocyte maturation. Coreceptor-associated Lck in DP thymocytes is dependent on CD45 to regulate phosphorylation of the negative regulatory C-terminal tyrosine (Y505), whereas regulation of Lck Y505 phosphorylation in DN thymocytes, and non-coreceptor-associated lck, is less dependent on CD45. These data suggest that the mechanisms that modulate the role of CD45 in regulating Lck activity are developmentally stage-specific. Furthermore, the present data identify a new role for coreceptor in maintaining and defining a pool of Lck that is regulated by CD45 and maintained in a primed active configuration before T cell activation. In addition, our data suggest that coreceptor-associated Lck may also be a preferential target for C-terminal tyrosine kinases like Csk, because this pool of Lck is more highly phosphorylated in the absence of CD45 than non-coreceptor-associated Lck. Thus, coreceptor-associated Lck may define a subset of Lck that is dynamically regulated by both CD45 and Csk, and in resting cells is biased toward a relatively hypophosphorylated state. Alterations in this balance after T cell activation or at different stages of T cell development may change the sensitivity and response of the T cell to both self and pathogen-derived peptides.

Previous phosphopeptide mapping studies demonstrating the importance of CD45 in regulating Lck Y505 phosphorylation have generally used CD45-deficient tumor cell lines, including CD4-negative variants of the BW thymoma cell line used in this study (7, 8, 9, 10, 11). Our observations that the profound hyperphosphorylation of Lck Y505 seen in the absence of CD45 expression was most pronounced in CD4+ cells had not been previously appreciated in these earlier reports using cells that do not express coreceptor. It should be noted, however, that the current data evaluating Lck Y505 phosphorylation in CD45-deficient BW cells that lack CD4, still demonstrate a 3- to 4-fold increase in phosphorylation when compared with the CD45+ counterpart similar to the previous studies (Fig. 3A, lanes 2 vs 4). In addition, in the absence of CD4, there is also a shift in Lck compartmentalization into lipid raft domains that may have been more efficiently solubilized in the earlier phosphopeptide mapping studies. As shown in Fig. 4B, this pool of Lck is hyperphosphorylated in the absence of CD45 independently of CD4 expression. In total, the data in the BW cells may be explained in part by decreased access of Lck to membrane-associated Csk in the nonraft fraction in CD4-negative cells, whereas CD4 expression promotes membrane localization of Lck in nonraft domains, where it may have greater access to both Csk and CD45. In the absence of CD4, there is a change in Lck membrane compartmentalization resulting in an increased fraction of Lck resident within lipid raft-like domains. This may also promote access to Csk, which is associated with raft-resident proteins like PAG/cbp.

Our results indicating that CD4-associated Lck is preferentially regulated by CD45, is similar to previous data from the laboratory of D. Alexander and colleagues (36), where the positive regulatory effect of CD45 on Lck kinase activity in a CD45-deficient tumor cell line was not detected in total cellular Lck, but was only evident upon immunoprecipitation of CD4-associated Lck. Our current data extend these observations by specifically examining the role of the coreceptor in affecting Lck Y505 phosphorylation in cell lines genetically reconstituted with various combinations of CD45 and CD4 and in biochemical coreceptor preclearing experiments from CD45-deficient cell lines and thymocytes. In addition, we have correlated changes in the expression of coreceptors during thymocyte development with changes in the role of CD45 in regulating Lck phosphorylation.

In contrast to BW cells, our data using differential detergent lysis of DN thymocytes suggests that CD45 does not play a major role in regulating non-coreceptor-associated Lck C-terminal tyrosine phosphorylation in both raft and nonraft domains. Although there is an increase in apparent raft-resident Lck in the DN thymocytes upon solubilization with maltoside, phosphorylation of this pool of Lck is much less affected by CD45 compared with both raft and nonraft Lck isolated from total thymocytes (Figs. 4A and 5). This may be due to the presence of another protein tyrosine phosphatase in DN cells that is active at this stage of development and compensates for the loss of CD45. However, the experiments described in Fig. 6 in which treatment of DN cells with the pan protein tyrosine phosphatase inhibitor pervanadate failed to induce significant Lck Y505 phosphorylation argue against this possibility. In addition, it is possible that in DN thymocytes Csk is less active, and thus the requirement for CD45 to counter Csk activity is reduced. Previous reports in Csk-deficient mice, however, suggest that Csk actively regulates Lck-dependent pre-TCR signaling (27). In RAG-deficient animals and in the absence of Csk, thymocytes develop to the DP stage apparently bypassing beta-selection and the requirement for a functional pre-TCR. These data suggest that Csk is indeed present and active at the DN stage of thymocyte development, and that additional CD45-independent mechanisms may be involved in promoting Lck activity in DN cells.

In addition to tyrosine phosphorylation-dependent alterations in Lck activity, Lck is also positively regulated by inter- and intramolecular interactions involving its Src homology 3 domain (37, 38, 39). These interactions may be particularly important during early thymocyte development when CD45 appears to have a more limited role. Indeed, a number of adaptor molecules are known to interact with Lck, in part due to SH3 domain-mediated interactions, and may play an important role in promoting Lck activity during early thymocyte development independently of CD45 (40, 41, 42, 43). The relative roles of these and other factors during pre-TCR signaling await further study.

Nevertheless, our data indicating differential roles for CD45 in regulating Lck Y505 phosphorylation during thymic development are consistent with the idea that regulation of pre-TCR signaling in DN thymocytes is intrinsically different from signaling in DP cells and mature T cells. It is thought that pre-TCR signaling in DN cells occurs autonomously in the absence of an extracellular ligand (44). This is in part facilitated by localization in lipid raft domains and by the tendency of pre-TCR{alpha}-chains to dimerize, and perhaps by an inherent difference in the sensitivity of DN cells to respond to low potency signals (33, 45, 46). Our current data indicate that in addition to the pre-TCR, Lck is also relatively enriched within lipid raft membrane microdomains in DN compared with Lck in total thymocytes. This difference in localization may promote Lck activity in the absence of CD45 by facilitating interaction with substrates and/or other positive regulators of Lck activity.

Changes in CD45 isoform expression during thymic development may also relate to the differences we have observed in the role of CD45 in regulating Lck Y505 phosphorylation during thymocyte development. A variety of mechanisms have been previously proposed to regulate CD45 protein tyrosine phosphatase activity, including serine phosphorylation, oxidation, and homodimerization (1, 47, 48, 49). These events may also be influenced by differential expression of CD45 isoforms. The regulated expression of distinct CD45 isoforms during T cell development and activation has suggested a role for the CD45 ectodomain in regulating both phosphatase activity and access of substrates to CD45. Cell lines transfected with distinct CD45 isoforms or variable size ectodomains, demonstrate differential ability to homodimerize, to associate with CD4 and/or the CD3/TCR complex, and exhibit differences in membrane microdomain compartmentalization (20, 21, 29, 31, 50). At the DN stage, thymocytes express a mixed pattern of mostly high m.w. CD45 isoforms, whereas DP cells predominantly express the low m.w. CD45RO isoform (51). Because CD45RO isoforms are less extensively glycosylated and seem to have enhanced access to coreceptor-associated lck, this is consistent with an increased role for CD45 at the DP stage. In contrast, the heavily glycosylated large m.w. CD45 isoforms may have relatively reduced access to lipid raft-associated Lck, and/or relatively diminished access to non-coreceptor-associated Lck, consistent with a reduced role for CD45 at the DN stage (20, 21, 29, 31).

In addition to factors that regulate the basal capacity or potential activity of CD45, CD45 protein tyrosine phosphatase activity and/or CD45 access to substrates may be further regulated following T cell activation (32, 52, 53, 54). These data suggest that the regulation of CD45 functional activity is likely to be complex and that different modes of regulation may be involved depending on the state of T cell activation and stage of development. Our current data indicate that CD45 substrates such as Lck may be compartmentalized in distinct pools and are regulated by both CD45-dependent and -independent mechanisms. Segregation of signaling molecules and/or macromolecular signaling complexes into distinct compartments may be a means to differentially regulate signaling pathways and outcomes of cell activation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Arthritis Foundation, American Cancer Society, and National Institutes of Health (Grant AI42963; to D.L.). Back

2 This work is in partial fulfillment of the requirements for a Ph.D. degree in Institute for Biomedical Sciences, George Washington University (for R.F.). Back

3 Address correspondence and reprint requests to Dr. David Leitenberg, Department of Microbiology, Immunology, and Tropical Medicine, George Washington University, 2300 I Street Northwest, Washington, DC 20037. E-mail address: dleit{at}gwu.edu Back

4 Abbreviations used in this paper: Csk, C-terminal Src kinase; DP, double positive; DN, double negative; NP-40, Nonidet P-40. Back

Received for publication July 28, 2006. Accepted for publication December 1, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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