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CD45 Function Is Regulated by an Acidic 19-Amino Acid Insert in Domain II That Serves as a Binding and Phosphoacceptor Site for Casein Kinase 2¹

Susanna F. Greer^2,*, Yan-ni Wang^2,*, Chander Raman and Louis B. Justement^3,*

^* Department of Microbiology, Division of Developmental and Clinical Immunology, and Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama, Birmingham, AL 35294

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 , ', or subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 and or ' and subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-aa insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Colocalization of the Src family protein tyrosine kinases (PTK)⁴ with the Ag receptor (AgR) complex on T and B cells plays an important role in the initiation and propagation of signal transduction in response to Ag stimulation (1, 2). Numerous studies have demonstrated that CD45, a transmembrane protein tyrosine phosphatase (PTP), also plays an important role in regulating lymphocyte biology (3, 4). CD45 expression is essential for normal T and B cell development and for optimal activation in response to AgR cross-linking (5, 6, 7, 8). It has been shown that B cell Ag receptor (BCR)-mediated signal transduction via Lyn, Fyn, and Blk is dependent on the expression of CD45 (9, 10, 11, 12). Similarly, in T cells, dephosphorylation of Lck and Fyn by CD45 leads to their activation and participation in TCR signaling cascades (13, 14, 15, 16). Thus, CD45 regulates reversible protein tyrosine phosphorylation, and therefore lymphocyte biology, through its ability to regulate Src PTK function.

Although significant progress has been made toward understanding how CD45 regulates AgR signaling, there is still much to be learned regarding the mechanism(s) by which CD45 substrate specificity and catalytic activity are controlled. The cytoplasmic tail of CD45 contains tandem repeat PTP domains, designated domain I (DI) and II (DII), that exhibit a significant degree of homology to PTP IB (40 and 33%, respectively) (4). It is apparent, however, that DI, but not DII, is catalytically active based on mutational analysis of conserved cysteine residues within the catalytic site of each. Results from in vitro as well as in vivo studies have shown that mutation of the conserved cysteine residue in DI to serine completely abrogates CD45 catalytic function, whereas mutation of the analogous cysteine residue in DII has no effect (17, 18, 19). Moreover, recent studies have shown that DI of CD45 exhibits phosphatase activity when expressed as a recombinant protein in the absence of DII, whereas DII has no phosphatase activity when isolated from DI (20).

Although DII does not directly play a role in dephosphorylation of substrates, there is substantial evidence to suggest that it may, in fact, influence both the catalytic activity and the substrate specificity of DI (18, 19, 20). Studies using recombinant CD45 have demonstrated that deletion of DII in its entirety can either decrease or completely abrogate the ability of DI to dephosphorylate artificial substrates in vitro. Smaller deletions within DII, including a truncation of the carboxyl-terminal 15 aa, affect the catalytic function of DI as well (18, 19). Finally, in vivo studies have demonstrated that replacement of CD45 DII with DII from the PTP LAR abrogates the ability of the chimeric CD45 molecule to reconstitute TCR-mediated IL-2 production due to an alteration in the ability to recruit TCR- (21).

DII of CD45 contains a unique 19-aa acidic insert that is not found in other transmembrane PTPs. Deletion of this insert has been shown to selectively alter the ability of CD45 to dephosphorylate artificial substrates in vitro (18, 19, 20). The 19-aa insert contains multiple casein kinase 2 (CK2) consensus sites, suggesting that the catalytic function of CD45 may be regulated by serine phosphorylation of DII. Indeed, studies have demonstrated that CD45 is phosphorylated by CK2 at a high stoichiometry (22) and that multiple residues within the 19-aa insert are phosphoacceptor sites for this kinase (23, 24). Phosphorylation of CD45 within the acidic insert has been shown to regulate both its substrate specificity as well as its activity in vitro (23, 24). Additionally, work has shown that decreased serine phosphorylation of CD45 is associated with a decrease in its catalytic function (24, 25). These findings demonstrate that the ability of DII to alter the substrate specificity and/or catalytic activity of DI resides in part within the unique 19-aa insert and is regulated by reversible serine phosphorylation (24, 25).

In this study experiments were conducted to further elucidate the physical/functional nature of the molecular interaction between CD45 and CK2. The results obtained demonstrate that CD45 and the / subunits of CK2 physically interact with one another via the 19-aa acidic insert in DII of CD45. Mutational analyses suggest that CD45 catalytic function is regulated by CK2-dependent binding to and/or phosphorylation of CD45 within the acidic insert.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and cell culture

The B lymphoma cell line K46-17 µm (K46), provided by Dr. M. Reth (Max Plank Institut fur Immunologie, Freiburg, Germany), was maintained in IMDM supplemented with 5% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 50 µM 2-ME, 100 µg/ml streptomycin-penicillin, and 50 µg/ml gentamicin (Sigma) at 37°C under 7% CO₂. The Jurkat human leukemic T cell line (clone E6-1) and the CD45-negative variant (J45.01) were purchased from American Type Culture Collection (Manassas, VA) and were maintained in RPMI 1640 supplemented as described above. To obtain thymocytes and splenocytes, 6- to 8-wk-old C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbor, ME) were sacrificed, and the thymi and spleens were removed. Single-cell suspensions were prepared, and the cells were resuspended in cold RPMI and then centrifuged at 1500 rpm for 5 min. Five milliliters of erythrocyte lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 10 µM Na₂EDTA, pH 7.4) was added to the cell pellet, and the cells were resuspended by vortexing and then centrifuged at 1500 rpm for 5 min. To isolate splenic B cells, splenocytes were washed in RPMI and resuspended in tissue culture supernatant containing the mAbs T24 and HO.13.4 (mouse anti-Thy1.2, 1 ml each) for 10 min on ice. Subsequently, low tox rabbit complement (Life Technologies, Grand Island, NY), 10 µg/ml DNase I (Sigma, St. Louis, MO), and 5 mM MgCl₂ were added, and the cells were incubated at 37°C for 40 min. The splenic B cells were washed in RPMI 1640 and resuspended in complete RPMI 1640 for use in experiments.

Biological reagents

The mAbs used in these studies were OKT3 (mouse IgG, anti-human CD3), I3/2.5 (rat IgG2b, anti-mouse CD45), 145.2-C11 (hamster, anti-mouse CD3), MB23G2 (rat IgG2a, anti-mouse CD45, B exon), MB4B4 (rat IgG2a, anti-mouse CD45, B exon), T24 (anti-mouse Thy 1.2), and HO.13–4 (anti-mouse Thy 1.2). The mAbs were purified using protein G-Sepharose 4B Fast Flow (Amersham-Pharmacia Biotech, Piscataway, NJ). Anti-human CD45 (mouse IgG2b, clone RPI-14; Upstate Biotechnology, Lake Placid, NY), and anti-human CK2 (mouse IgM, clone 10; Transduction Laboratories, Lexington, KY) mAbs were purchased for these studies. Rabbit antiserum specific for CK2 was obtained from Dr. D. W. Litchfield (Department of Biochemistry, University of Western Ontario, London, Canada). Polyclonal goat anti-rabbit IgG coupled to HRP, goat anti-mouse IgG coupled to HRP, and streptavidin coupled to HRP were purchased from BioSource (Camarillo, CA). Protein A-agarose was obtained from Life Technologies.

Immunoprecipitation and immunoblotting

K46 B cells or Jurkat T cells (2 x 10⁷/sample) were resuspended in 1 ml of RPMI containing 5% FBS and rested at 37°C for 20 min. The cells were stimulated with mAb directed against either the BCR (anti-IgM, B76, 10 µg/ml) or CD3 (OKT3, 10 µg/ml) at 37°C for 10 min. Control samples received neither anti-BCR nor anti-CD3 mAbs, but were incubated at 37°C for 10 min. Reactions were stopped by the addition of ice-cold PBS. Next, cells were washed twice with ice-cold PBS and lysed in 0.5 ml lysis buffer (25 mM HEPES (pH 7.8), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, and 0.1 mM Na₃VO₄) containing 1% Nonidet P-40. Cell lysates were incubated for 1 h on ice and then centrifuged at 13,000 x g for 15 min at 4°C. Detergent-soluble lysates were precleared by incubation with protein G-Sepharose and protein A-agarose for 1 h at 4°C. CD45 was immunoprecipitated from precleared K46 lysates by the addition of I3/2.5 mAb (10 µg/ml) plus protein G-Sepharose for 1 h at 4°C with rotation. CD45 was immunoprecipitated from precleared Jurkat lysates by incubation with precoated protein G-Sepharose beads for 1 h at 4°C with rotation. For these experiments protein G-Sepharose beads were precoated initially with the anti-human CD45 mAb RPI-14 and then with saturating amounts of mAb I3/2.5 (anti-murine CD45). Immune complexes bound to beads were collected and washed five times with lysis buffer containing 0.2% Nonidet P-40. The beads were resuspended in 25 µl SDS-PAGE sample-reducing buffer, boiled for 4 min, and centrifuged at 15,000 x g for 5 min. After centrifugation, 20 µl supernatant from each immune complex sample and 5 µl from total lysate control samples were separated on 8–10% acrylamide gels by SDS-PAGE and transferred to Hybond-ECL nitrocellulose membranes (Amersham-Pharmacia Biotech). The membranes were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and were washed five times with TBST. The membranes were then incubated with anti-CK2 mAb or the polyclonal antiserum against CK2 for 1 h at room temperature and were washed five times with TBST. Next, the membranes were incubated with the appropriate secondary Ab coupled to HRP for 1 h at room temperature and washed five times with TBST. The CK2 band was visualized using ECL with Supersignal reagent (Pierce, Rockford, IL).

In parallel experiments thymocytes (2 x 10⁷/sample) were resuspended in complete RPMI containing 5% FBS and rested at 37°C for 20 min. The cells were stimulated with mAb directed against CD3 (145.2-C11, 10 µg/ml) for 10 min at 37°C. Control samples received no stimulation, but were incubated at 37°C for 10 min. Reactions were stopped by the addition of ice-cold PBS, the cells were lysed as described above, and the lysates were precleared with protein G-Sepharose and protein A-agarose. CD45 was immunoprecipitated using protein G-Sepharose beads that had been precoated first with the mAbs MB23G2 and MB4B4 (anti-murine CD45 B exon) and then with saturating amounts of the I3/2.5 mAb. Similarly, splenic B cells (2 x 10⁷/sample) were resuspended in complete RPMI containing 5% FBS and were rested at 37°C for 20 min. The cells were stimulated with mAb directed against the BCR as described above. Control samples received no stimulation, but were incubated for 10 min at 37°C. Reactions were stopped by the addition of ice-cold PBS, the cells were lysed, and the lysates were precleared with protein G-Sepharose and protein A-agarose. CD45 was immunoprecipitated with protein G-Sepharose beads precoated with a saturating amount of the mAb I3/2.5. Samples were analyzed by Western blotting as described above.

Analysis of CK2 association with wild-type CD45 and CD45 mutants transfected into J45.01 cells was performed using the solid phase immunoprecipitation technique. Ninety-six-well microtiter plates were coated with I3/2.3 (25 µg/ml in 100 µl) in PBS at 4°C overnight, after which the plates were washed five times with PBS. J45.01 transfectants expressing wild-type CD45 or mutant CD45 (2 x 10⁷/sample) were lysed in 150 µl lysis buffer containing 1% Nonidet P-40 for 1 h on ice. The lysates were centrifuged at 13,000 x g for 15 min, and the detergent-soluble material (100 µl) was added to the wells of the precoated 96-well microtiter plate. The plates were incubated at 4°C overnight, then washed three times, and 38 µl SDS-PAGE sample buffer was added. The plates were incubated at 70°C for 20 min, and the sample buffer was mixed in the wells and transferred to 1.5-ml microfuge tubes. The samples were boiled for 5 min, and the immune complexes were separated by SDS-PAGE on 10% acrylamide gels. Coprecipitation of CK2 with CD45 was detected by Western blotting as described previously.

Yeast two-hybrid assay

To generate the GAL4 binding domain-CD45 cytoplasmic domain (BD-CD45) fusion, the entire 702-aa cytoplasmic domain of CD45 (Tyr⁵⁶⁴–Thr¹²⁶⁸) was amplified from the CD45 minigene/ECMVneo cDNA construct (a gift from Dr. M. Thomas, Washington University, St. Louis, MO) using PCR with sense (5'-TATAAAATCTATGATCTGCGC-3') and antisense (5'-TGTGTTCACCTTTGCCACTG-3') primers. The PCR product encoding the cytoplasmic domain of CD45 was directionally cloned into the yeast expression vector pGBT9 (Clontech, Palo Alto, CA) and analyzed using fluorescent dye terminator sequencing (ABI PRISM; Perkin-Elmer, Branchburg, NJ) to confirm sequence accuracy. The sequenced BD-CD45 fusion construct was transformed into MAV203 yeast cells according to the manufacturer’s instructions (ProQuest Two-Hybrid System; Life Technologies), and the yeast was screened to rule out nonspecific activation of the GAL4 promoter. The BD-CD45 construct was then used in yeast two-hybrid screens to assay for interactions between the GAL4 BD-CD45 and GAL4 AD-CK2 fusion proteins encoded by the AD-CK2, AD-CK2', and AD-CK2 constructs that had previously been generated using the yeast expression vector pACTII (Matchmaker, Clontech). Growth on uracil-deficient plates was assessed at 48 h following transfer of cotransformed yeast from tryptophan-deficient (Trp^-) and leucine-deficient (Leu^-) selection medium.

CD45 constructs for yeast three-hybrid analysis

To generate the GAL4 binding domain-CD45 DI fusion construct (BD-CD45 PTPDI), the QuikChange mutagenesis kit from Stratagene (La Jolla, CA) was used as directed by the manufacturer to introduce a premature stop codon after Thr⁹³⁰ in the BD-CD45 construct using the primers 5'-GAGTTGGAGGACATAGCACACATTGG-3' (sense) and 5'-CCAATGTGCTATGTCCTCCAACTC-3' (antisense; Fig. 1). The GAL4 binding domain-CD45 DII fusion construct (BD-CD45 PTPDII) was generated by PCR-mediated amplification of CD45 PTP DII (Ser⁹⁰³–Thr¹²⁶⁸) from the CD45 minigene/ECMVneo cDNA construct using the primers 5'-GTGACCCCTCCCCTCTGG-3' (sense) and 5'-TGTGTTCACCTTTGCCACTG-3' (antisense). The PCR product encoding CD45 PTP DII was then directionally cloned into the yeast expression vector pGBT9. To generate the BD-CD45958–973 and the BD-CD45 PTPDII958–973 fusion constructs, the QuikChange mutagenesis kit was used with primers 5'-CCACTTAAGCATGAACTGGAGATGGACTCAGAAGAAACCAGC-3' (sense) and 5'-GCTGGTTTCTTCTGAGTCCATCTCCAGTTCATGCTTAAGTGG-3' (antisense). All constructs were sequenced to ensure the absence of PCR-induced artifacts and the presence of the desired mutations and/or deletions by bidirectional nucleotide sequencing using dye terminator chemistry (ABI PRISM).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Schematic representation of BD-CD45 cytoplasmic domain fusion protein constructs. The full-length cytoplasmic domain of CD45 and PTPDII was amplified from a CD45 minigene construct by PCR and were directionally subcloned into the pGBT9 vector. All other mutations of the CD45 cytoplasmic domain were introduced into the initial BD-CD45 construct as described in Materials and Methods.

To generate the third construct necessary for the three-hybrid assay, CK2 was amplified from the AD-CK2 construct by PCR with sense (5'-AGTGGATCCGCTGACGTGAAGATGAGCA-3') and antisense (5'-AGTGTCGACTCCTGAAAGGGTGGCAAAAA-3') primers. CK2 was cloned into the yeast expression vector p14 MET25 (a gift from Dr. K. S. Campbell, Fox Chase Cancer Center, Philadelphia, PA) and transformed into MAV203 yeast cells. The p14 MET25-CK2 construct was then screened for nonspecific activation of the GAL4 promoter. Yeasts that contained the p14 MET25-CK2 plasmid were used in yeast three-hybrid screens in which they were cotransformed with AD-CK2 and BD-CD45, AD-CK2' and BD-CD45, AD-CK2 and BD-CD45 PTPDI, AD-CK2' and BD-CD45 PTPDI, AD-CK2 and BD-CD45 PTPDII, AD-CK2' and BD-CD45 PTPDII, AD-CK2 and BD-CD45958–973, AD-CK2' and BD-CD45958–973, AD-CK2 and BD-CD45 PTPDII958–973, and AD-CK2' and BD-CD45 PTPDII958–973. Growth on uracil-deficient plates was assessed at 48 h following transfer of yeast from Trp^-, Leu^-, and histidine-deficient (His^-) selection medium.

Reconstitution of CD45 expression in the J45.01 CD45-negative Jurkat cell line

The J45.01 CD45-negative cell line was used for structure/function studies of CD45 following electroporation with cDNA encoding wild-type or mutated forms of this PTP. The CD45 minigene expression constructs used for electroporation were provided by Dr. M. Thomas (Washington University). Two expression constructs were used; the CD45 minigene construct encodes the high m.w. isoform of mouse CD45 (CD45RA), and the CD45 minigene construct encodes the low m.w. isoform of mouse CD45 (CD45RO). The CD45 and - minigene constructs were mutagenized using the QuikChange mutagenesis kit from Stratagene to delete the acidic insert in DII (aa 958–973 and 808–826, respectively) resulting in the generation of cDNA minigene constructs encoding CD45RA: 958–973 and CD45RO:808–826. Additional mutations were introduced into the CD45RA minigene construct, resulting in conversion of the serines contained within the acidic insert in DII (S965, 968, 969, and 973) to alanine (CD45RA:S965/968/969/973A). Electroporation was used to introduce the wild-type and mutant CD45 minigene constructs into J45.01 cells. J45.01 cells (1 x 10⁷) were resuspended in 500 µl IMDM and were transfected with 10 µg of cDNA using a Becton Dickinson electroporator (San Jose, CA) with settings of 960 mF and 0.25 kV. After 48 h cells were selected in medium containing 1 mg/ml G418 (Life Technologies). Drug-resistant cells were analyzed by flow cytometry after staining with biotinylated I3/2.3 and PE-streptavidin to determine the relative surface expression of mouse CD45. Multiple rounds of FACS were used to isolate bulk populations of transfected J45.01 cells that expressed comparable levels of wild-type and mutant CD45.

Measurement of calcium mobilization

Studies were performed with parental Jurkat cells (clone E6-1), J45.01 CD45-negative cells, and J45.01 transfectants in which Ca²⁺ mobilization was assayed in response to CD3 cross-linking as described previously (26). Cells were loaded with the Ca²⁺ indicator dye indo-1/AM (Molecular Probes, Eugene, OR) at a final concentration of 5 µM. Cells loaded with indo-1 were analyzed using a Becton Dickinson FACSVantage flow cytometer equipped with an Enterprise laser from Coherent (Santa Clara, CA) set for excitation at approximately 364 nm at a power setting of 60 mW. Fluorescence emissions were separated by a 505-nm short pass beam splitter into two component emissions by passage through 405- and 485-nm centered 10-nm bandpass filters to detect violet and blue, respectively. The ratio of emissions was calculated, and a plot was constructed of fluorescence ratio vs time. Indo-1-loaded cells (1 x 10⁶/sample) were analyzed by flow cytometry to establish a baseline for the concentration of free intracellular Ca²⁺. Once the baseline measurement had been taken, the analysis was stopped, and the cells were stimulated by the addition of anti-CD3 mAb (OKT3, 0.1–10 µg/ml), after which the analysis was immediately resumed. To ensure that all cell lines were loaded equivalently with indo-1, the intracellular concentration of free Ca²⁺ was monitored for cells incubated in the presence of ionomycin (1 µM final concentration).

Measurement of mitogen-activated protein kinase (MAPK) activation

Experiments were performed with parental Jurkat cells, J45.01 CD45-negative cells, and J45.01 transfectants to monitor CD3-mediated activation of the MAPK extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (Jnk). Cells (1 x 10⁷/sample) were resuspended in 1 ml RPMI containing 5% FBS and were rested at 37°C for 20 min. Subsequently, the cells were stimulated with OKT3 (10 µg/ml) or PMA (100 ng/ml) for the period of time indicated, or they were left untreated. The reactions were stopped by the addition of ice-cold PBS, and the cells were washed twice and then lysed in 0.5 ml lysis buffer containing 1% Nonidet P-40. Cell lysates were incubated on ice for 1 h and then centrifuged at 13,000 x g for 15 min at 4°C. Detergent-soluble material (25 µl) was mixed with an equal volume of 2x SDS-PAGE sample reducing buffer, boiled, and centrifuged at 15,000 x g for 5 min. Twenty microliters from each sample were separated by SDS-PAGE on 10% acrylamide gels and transferred to Hybond-ECL nitrocellulose. The membranes were blocked and then incubated with either anti-phospho-Erk1/2 (Thr²⁰², Tyr²⁰⁴) or anti-phospho-Jnk (Thr¹⁸³, Tyr¹⁸⁵) polyclonal Ab (New England Biolabs, Beverly, MA). Next, the membranes were washed and probed with goat anti-rabbit IgG coupled to HRP (BioSource). Phosphorylation of Erk1/2 and Jnk was visualized using ECL. To ensure equal loading of Erk1/2 or Jnk, the membranes were stripped by incubating them in buffer containing 10 mM Tris, pH 2.3, and 150 mM NaCl at 70°C for 1 h, after which they were washed extensively in TBST. The membranes were then blocked and probed with anti-Erk1/2 or anti-Jnk polyclonal Ab to detect the total amount of Erk1/2 or Jnk, respectively. The proteins were visualized by incubating the membranes with goat anti-rabbit IgG coupled to HRP, followed by ECL.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CK2 associates with CD45 in B and T cell lines

Previous studies have demonstrated that changes in the phosphorylation status of CD45 play a role in regulating its substrate specificity and/or catalytic activity. It has been shown that tyrosine phosphorylation of CD45 followed by CK2-dependent serine phosphorylation results in increased activity based on dephosphorylation of artificial substrates in vitro (23). More recently, work characterizing the phosphorylation of CD45 by CK2 has demonstrated that DII of CD45 contains characteristic CK2 phosphorylation sites within a 19-aa insert and that phosphorylation of those sites leads to an increase in the maximum velocity of CD45 in vitro (24). To further elucidate the nature of the physical interaction between CD45 and CK2 in lymphocytes, coimmunoprecipitation experiments were performed. Jurkat cells were incubated in medium alone or were stimulated with anti-CD3 mAb (OKT3) followed by immunoprecipitation of CD45 from detergent-soluble lysates. Western blot analysis of the CD45 immune complex with mAb specific for CK2 revealed that CK2 coprecipitates with CD45 from unstimulated cells and that upon stimulation, the amount of CK2 associated with CD45 increases compared with that in the unstimulated sample (Fig. 2A). Samples from unstimulated detergent-soluble Jurkat lysates incubated with protein G-Sepharose alone and probed with anti-CK2 did not contain CK2. To determine whether a similar physical association occurs between CD45 and CK2 in B cells, coimmunoprecipitation experiments with the K46 B lymphoma cell line were performed. K46 cells were incubated in medium alone or were stimulated with anti-BCR mAb (B76), after which CD45 was immunoprecipitated from detergent-soluble lysates. Western blot analysis of the CD45 immunoprecipitates revealed, similar to T cells, that CK2 coprecipitates with CD45 from unstimulated B cells and that the amount of associated CK2 increases upon AgR stimulation (Fig. 2B). As before, samples from detergent-soluble K46 lysates incubated with protein G-Sepharose alone and probed with anti-CK2 mAb did not contain detectable amounts of CK2. These findings suggest that CD45 and CK2 interact with one another in a constitutive manner in B and T cells and that upon stimulation through the AgR, the degree to which they interact increases. Western blotting could not be performed to monitor the recovery of CD45 due to a lack of anti-CD45 Abs that could be used for blotting. Nevertheless, control experiments to ensure that cellular activation does not affect recovery of CD45 were performed in which CD45 immune complexes were biotinylated before separation by SDS-PAGE. Loading of CD45 was detected by probing nitrocellulose membranes with streptavidin coupled to HRP, after which ECL was used to visualize CD45 on the membrane. Equivalent recovery of CD45 from both T and B cell lysates was consistently observed regardless of whether the cells had been stimulated (data not shown). In conclusion, these data provide the first evidence of a physical interaction between CD45 and the serine/threonine kinase CK2.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. CK2 is physically associated with CD45 isolated from T and B cell lines. A, Jurkat cells (2 x 107/sample) were incubated in medium alone (NT, 10 min) or in the presence of anti-CD3 (CD3) mAb (OKT3, 10 µg/ml) for 1–10 min at 37°C. The cells were lysed, and CD45 was immunoprecipitated (I.P.) using the mAb RPI-14 plus protein G-Sepharose. Western blotting was performed to detect CK2 using a rabbit polyclonal antiserum followed by the addition of goat anti-rabbit IgG coupled to HRP. The presence of CK2 was visualized using ECL. Whole cell lysates were run as a positive control for the detection of CK2. Protein G-Sepharose alone was incubated with lysates to control for nonspecific adsorption of CK2 to the bead matrix (lane 1). B, K46 B lymphoma cells (2 x 107/sample) were incubated in medium alone (10 min) or in the presence of anti-IgM (Ig) mAb (B76, 10 µg/ml) for 1–10 min. CD45 was immunoprecipitated from cell lysates using the anti-CD45 mAb I3/2.5 and protein G-Sepharose. The presence of CK2 in CD45 immune complex material was assayed by Western blotting using an anti-CK2 mAb with a secondary goat anti-mouse IgG Ab coupled to HRP. CK2 was visualized using ECL. Controls were run similar to those described for the Jurkat experiment.

To determine whether the physical association between CD45 and CK2 demonstrated in T and B cell lines also occurs in nontransformed cells, thymocytes were incubated in medium alone or were stimulated through the TCR complex with anti-CD3 mAb (145.2-C11). CD45 was immunoprecipitated from detergent-soluble lysates, and Western blotting with antiserum specific for CK2 was performed to detect the presence of CK2. As shown in Fig. 3, CK2 associated with CD45 in thymocytes. Although the data clearly show the constitutive nature of the interaction, as seen in the unstimulated sample, CD45 and CK2 exhibited an enhanced interaction with one another in response to CD3 cross-linking. Splenic B cells were also incubated in medium alone or were stimulated through the BCR with anti-IgM mAb (B76). CD45 was immunoprecipitated from detergent-soluble lysates, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were probed with antiserum specific for the subunit of CK2, and as shown in Fig. 3, a low level basal association between CK2 and CD45 could be seen that was enhanced upon cross-linking of the BCR complex. In both thymocyte and B cell experiments, protein G-Sepharose alone was incubated with cell lysates to demonstrate the specificity of the CD45/CK2 interaction. As described previously, recovery and loading of CD45 were monitored in selected experiments based on biotinylation of CD45 immune complex material and Western blotting using streptavidin coupled to HRP (data not shown). These studies clearly show that the serine/threonine kinase CK2 interacts with CD45 in nontransformed T and B cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. CK2 is physically associated with CD45 isolated from thymocytes and splenic B cells. Either thymocytes or splenic B cells (2 x 107) were incubated in medium alone (10 min) or in the presence of anti-CD3 (CD3, 145.2C-11, 10 µg/ml, 10 min) or anti-IgM (mIg, B76, 10 µg/ml, 10 min), respectively, before lysis in 1% Nonidet P-40. CD45 was immunoprecipitated (I.P.) using the mAbs I3/2.5, MB23G2, or MB4B4 in conjunction with protein G-Sepharose. The presence of CK2 in CD45 immune complexes was assayed by Western blotting using either polyclonal anti-CK2 antiserum (thymocytes) or anti-CK2 mAb (splenic B cells), followed by addition of the appropriate secondary Ab coupled to HRP. CK2 was visualized using ECL. Whole cell lysate was run as a positive control for CK2, and protein G-Sepharose alone was added to lysates to control for nonspecific adsorption of CK2 (lanes 1 and 5).

The yeast two-hybrid assay was used to map the sites of physical interaction between the cytoplasmic domains of CD45 and CK2. The CK2 holoenzyme is a tetrameric protein consisting of two interchangeable catalytic subunits, (45 kDa) and ' (40 kDa), and two regulatory subunits (26 kDa each) arranged in one of the following configurations: ₂₂, '₂₂, or '₂ (27, 28). Studies have demonstrated that CK2 can interact with its substrates as a holoenzyme or via either the catalytic /' or regulatory subunit. Previous studies have demonstrated that CK2' is the primary subunit responsible for phosphorylating CD45 in vitro, and that the CK2 subunit was not necessary for CD45 phosphorylation (24). Based on these observations we used the AD-CK2, AD-CK2', and AD-CK2 GAL4 activation domain-CK2 fusion constructs to determine whether individual CK2 subunits exhibit the ability to interact with the cytoplasmic domain of CD45 in the yeast two-hybrid assay. The BD-CD45 fusion construct was generated by amplifying the entire 702-aa cytoplasmic domain of CD45 (Tyr⁵⁶⁴-Thr¹²⁶⁸) and subcloning it in-frame into the GAL4 binding domain fusion vector pGBT9. Yeast were cotransformed with BD-CD45, and each of the GAL4 activation domain-CK2 fusion constructs. None of the CK2 subunits was observed to interact with the cytoplasmic domain of CD45 in the yeast two-hybrid assay as assessed by growth on uracil-deficient selection medium (Table I).

Association between CK2 and CD45 is mediated by the unique 19-aa acidic insert in PTP DII

Because CD45 contains tandem repeat PTP domains, and each of these domains contains consensus CK2 phosphorylation sites, it was of interest to determine whether CK2 interacts with both DI and DII or selectively with only one domain. To address this question, the yeast three-hybrid assay was used once again in conjunction with GAL4 binding domain fusion protein constructs that encoded either DI or DII of CD45. The ability of the full-length CD45 cytoplasmic domain (BD-CD45), CD45 PTP DI alone (BD-CD45 PTPDI), or CD45 PTP DII alone (BD-CD45 PTPDII) to interact with AD-CK2 + CK2 or AD-CK2' + CK2 was assessed based on the growth of cotransformed yeast on uracil-deficient selection medium (Fig. 4). Yeast containing BD-CD45 + AD-CK2 + CK2, or BD-CD45 + AD-CK2' + CK2 grew in the absence of uracil as previously described. Yeast containing BD-CD45 PTPDII + AD-CK2 + CK2 or BD-CD45 PTPDII + AD-CK2' + CK2 also grew in the absence of uracil. It should be noted that the growth characteristics of yeast were identical regardless of whether the BD-CD45 full-length construct or the BD-CD45 PTPDII mutant was used for transformation. In contrast, yeast cotransformed with BD-CD45 PTPDI + AD-CK2 or ' + CK2 did not grow in the absence of uracil (Fig. 4). These results clearly demonstrate that PTP DII, but not DI, mediates the interaction between CK2 and CD45. These results further suggest that the ability of DII to interact with CK2 may be due to the presence of one or more unique motifs in DII that are not contained within DI.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. CK2 selectively interacts with PTP DII of CD45. The yeast three-hybrid assay was used to determine whether CK2 selectively interacts with either DI or DII of CD45, or with both domains. A, The MAV203 strain of yeast was cotransformed with vectors encoding BD-CD45, BD-CD45 PTPDI, or BD-CD45 PTPDII in conjunction with CK2 and AD-CK2/'. Transformed yeast were plated on dropout medium (Trp-, Leu-, His-) to select for those cells that had taken up all three vectors. These yeast were then plated on uracil-deficient medium and incubated at 30°C for 48 h to assay for the interaction between CD45 and CK2. B, PTP DII of CD45 interacts with both CK2 and CK2' in the presence of CK2. The photograph depicts the growth of equivalent numbers of yeast cells platted on dropout medium in the presence or the absence of uracil for 48 h at 30°C.

Structure/function experiments were conducted using the CD45-negative Jurkat cell line J45.01 (29, 30). Compared with CD45-positive parental Jurkat cells (clone E6-1), these cells exhibit a complete lack of responsiveness to stimulation through CD3 due to the loss of CD45 expression (Fig. 5). However, when the J45.01 cell line is transfected with minigene expression constructs encoding either wild-type mouse CD45RA or CD45RO, it is possible to reconstitute full responsiveness to ligands that cross-link the TCR complex. As shown in Fig. 5, stimulation of the J45.CD45RA and J45.CD45RO transfectant cell lines with anti-CD3 mAb resulted in a Ca²⁺ mobilization response comparable to that observed for parental Jurkat cells. Thus, expression of either mouse CD45RA or CD45RO is sufficient to restore the cell’s responsiveness, presumably through the maintenance of a pool of active Src family PTKs that transduce a signal in response to TCR complex ligation. To assess the importance of the DII acidic motif in the regulation of CD45 function, the CD45RA and CD45RO minigene constructs were mutagenized, resulting in deletion of the 19-aa acidic insert in DII. After transfection with these constructs, J45.01 cells that expressed the mutant forms of CD45RA and CD45RO were selected based on immunofluorescence staining and cell sorting to isolate nonclonal transfectant cell lines. J45.CD45RA:958–973 and J45.CD45RO:808–826 cell lines that express comparable levels of CD45 compared with J45.CD45RA and J45.CD45RO transfectants (data not shown) were analyzed to assess CD3-dependent signal transduction. As shown in Fig. 6, deletion of the 19-aa insert affects the function of both CD45RA and CD45RO based on changes in the Ca²⁺ mobilization response detected in cells treated with anti-CD3 mAb. The results depicted in Fig. 6 are representative of nine independent experiments with the J45.CD45RA:958–973 transfectants and five experiments with the J45.CD45RO:808–826 transfectants. Importantly, it was observed that deletion of the 19-aa insert had a much greater effect on the ability of CD45RO to reconstitute Ca²⁺ mobilization as opposed to CD45RA (Fig. 6). Whereas deletion of the acidic insert causes a change in the kinetics, but not the final magnitude, of the Ca²⁺ mobilization response in cells transfected with CD45RA, the same mutation results in a significant decrease in the magnitude of the Ca²⁺ flux in cells that express CD45RO. This finding suggests that the function of specific CD45 isoforms may be differentially controlled by CK2-dependent post-translational modification.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Restoration of CD3-mediated signal transduction by expression of murine CD45 in the human Jurkat T cell line. CD45-deficient Jurkat cells (J45.01 clone) were transfected with CD45 minigene constructs encoding wild-type mouse CD45RA or CD45RO. Immunofluorescence staining was used to assess CD45 expression, and cell sorting was used to isolate bulk populations of cells that expressed comparable levels of CD45. Jurkat transfectants (1 x 106/ml/sample) were loaded with indo-1/AM at a final concentration of 5 µM. The basal concentration of free intracellular Ca2+ was measured, after which anti-CD3 mAb (OKT3, 1 µg/ml) was added to the sample, and the analysis resumed. Indo-1 loading was assessed by stimulating cells with ionomycin (data not shown). The results are representative of six independent experiments.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. Deletion of the 19-aa insert in DII alters CD3-mediated Ca2+ mobilization. Transfectant J45.01 Jurkat cell lines expressing comparable levels of wild-type or mutant CD45RA or CD45RO were loaded with indo-1/AM at a final concentration of 5 µM. For each transfectant cell line, baseline Ca2+ measurements were taken, after which the cells were stimulated with anti-CD3 mAb (OKT3, 1 µg/ml), and the analysis was immediately resumed. Indo-1 loading was assessed for each cell line using ionomycin (data not shown). The results depicted are representative of seven independent experiments for J45.CD45RA and J45.CD45RA:958–973, and three independent experiments for J45.CD45RO and CD45RO:808–826. FL, Fluorescence.

View larger version (62K): [in this window] [in a new window]   FIGURE 7. Deletion of the 19-aa insert in DII alters CD3-mediated activation of MAP kinases. Transfectant J45.01 Jurkat cell lines expressing comparable levels of wild-type or mutant CD45RA or CD45RO were incubated in RPMI with 5% FBS in the presence or the absence of OKT3 (10 µg/ml) or PMA (100 ng/ml) for the times indicated. Cells were incubated in medium alone for 10 min (NT). A, Lysates from parental Jurkat cells and J45.01 transfectants expressing wild-type CD45RA or CD45RA lacking the acidic insert were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with phospho-specific Ab to detect activated Erk1/2 (top). The membrane was stripped and reprobed with Ab directed against Erk1/2 to detect the total amount of kinase loaded in each lane (bottom). B, Membranes were probed with phospho-specific Ab to detect activated Jnk (top). Membranes were stripped and reprobed with anti ()-Jnk Ab to detect the total amount of Jnk loaded in each lane (bottom).

Results obtained from yeast three-hybrid analyses demonstrated that the 19-aa insert in DII is important for the physical association between CD45 and CK2. Thus, it was logical to conclude that the suboptimal reconstitution of CD3-mediated signaling observed in the J45.CD45RA:958–973 and J45.CD45RO:808–826 cell lines is due to the inability of CK2 to associate with CD45. However, because deletion of the 19-aa insert inherently removes the serine residues that are phosphorylated by CK2 (24), it was not possible to elucidate the relative importance of the association between CD45 and CK2 vs phosphorylation of CD45 by CK2. Therefore, additional mutations were introduced into the CD45RA minigene construct, resulting in the conversion of four serine residues contained within the DII acidic insert (S965, 968, 969, and 973) to alanine. J45.CD45RA:S965/968/969/973A transfectants were isolated that expressed comparable levels of CD45 compared with J45.CD45RA and J45.CD45RA:958–973 based on immunofluorescence staining and cell sorting (data not shown). Analysis of Ca²⁺ mobilization in response to CD3 cross-linking revealed that mutation of the four serine residues to alanine resulted in a shift of the Ca²⁺ mobilization response comparable to that observed in the J45.CD45RA:958–973 transfectant cell line (Fig. 8).

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Mutation of serine residues 965, 968, 969, and 973 within the acidic insert of CD45RA alters CD45 function. J45.01 transfectants expressing comparable levels of wild-type and mutant CD45RA (CD45RA:965/968/969/973) were loaded with indo-1/AM at a final concentration of 5 µM. The basal level of free intracellular Ca2+ was measured for each cell line before stimulation with anti-CD3 mAb (OKT3, 1 µg/ml). Treatment of cells with ionomycin (1 µM) was used to ensure equal loading of indo-1 (data not shown). The results depicted are representative of three independent experiments. FL, Fluorescence.

View larger version (52K): [in this window] [in a new window]   FIGURE 9. Deletion of the acidic insert in PTPDII of CD45 abrogates the physical interaction with CK2 in J45.01 transfectants. CD45-negative Jurkat (J45.01), and J45.01 transfectants expressing either wild-type or mutant CD45 (1 x 107/sample) were lysed in buffer containing 1% Nonidet P-40. The cleared lysates were added to a 96-well microtiter plate precoated with anti-CD45 mAb (I3/2.3, 25 µg/ml), and the plate was incubated at 4°C overnight. The plate was washed, and SDS-PAGE sample buffer was added, after which the plate was incubated at 70°C. The plate-bound material was resolved by SDS-PAGE and transferred to nitrocellulose. Western blotting was performed to detect CK2 that coprecipitated with CD45. Whole cell lysate from J45.01 cells was run as a positive control (lane 1). I.P., immunoprecipitated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study the nature of the interaction between CD45 and CK2 was further investigated using yeast two-hybrid analysis, demonstrating that the individual , ', or subunits of CK2 do not associate with CD45. This finding is in contrast to previous work demonstrating that individual subunits of CK2 exhibit the ability to interact with a large number of substrates and/or regulatory proteins. For example, the CK2 subunit alone interacts with PP2A, c-Abl, nucleolin, and insulin receptor substrate 1 (36, 37, 38), whereas CK2 has been shown to interact with the serine/threonine kinase Mos (39) and the cell surface receptor CD5 (40). Although it was formally possible that the lack of a detectable interaction between the individual subunits of CK2 and CD45 in the yeast two-hybrid assay could be due to the requirement for an intermediate protein that physically couples CD45 and CK2, this hypothesis was not supported by the results from yeast three-hybrid analysis. The yeast three-hybrid assay revealed that the physical interaction between CD45 and CK2 requires the presence of both the or ' and subunits of CK2. It is interesting to note that previous studies have shown that either the CK2 or ' subunit is sufficient to mediate phosphorylation of CD45 in vitro in the absence of the subunit. Although the subunit may not be required for phosphorylation of CD45 in vitro, the results obtained in this study demonstrate that it is important for the physical interaction between CD45 and CK2. Thus, the intact CK2 holoenzyme may be required for recruitment and binding to CD45, which presumably lead to phosphorylation of the acidic 19-aa insert in DII.

CD45 is unique among the transmembrane tandem repeat PTPs in that DII contains an acidic 19-aa insert. This insert is highly conserved among all species and contains within it four CK2 consensus phosphorylation sites (Ser⁹⁶⁵, Ser⁹⁶⁸, Ser⁹⁶⁹, and Ser⁹⁷³ in CD45RA) (41). Indeed, studies have demonstrated that these residues are phosphorylated by CK2 leading to a 3-fold increase in the maximum velocity of CD45 and that the increase in CD45 catalytic activity can be reversed by treatment with the phosphatase PP2A (24). Mapping studies performed using the yeast three-hybrid assay revealed that the binding site for CK2 is also located within the 19-aa insert (residues 958–973 in CD45RA). Although a detailed analysis of the specific residues involved in binding of CK2 was not performed, it is likely that the interaction involves residues surrounding the four serines in the insert because both the /' and subunits are required. Presumably, the / subunits interact with one or more residues that flank the conserved serines, leaving these critical residues available for phosphorylation by the or ' subunits. Further evaluation of the specific residues important for CK2 binding will require scanning alanine mutagenesis of the 19-aa insert in conjunction with the yeast three-hybrid system. Nevertheless, it is apparent that the unique insert in DII of CD45 is required for binding of CK2, which presumably facilitates phosphorylation of CD45 by this serine/threonine kinase.

The functional importance of the 19-aa insert in CD45 was demonstrated by mutational studies in which CD45RA and CD45RO isoforms lacking this insert were expressed in CD45-deficient Jurkat cells. The results demonstrate that deletion of the acidic insert alters the kinetics of the CD3-mediated Ca²⁺ mobilization response in Jurkat transfectants that express the CD45RA:958–973 isoform. The equivalent mutation in CD45RO (CD45RO:808–826) has a much more significant effect on the ability of this isoform to reconstitute signaling via CD3 compared with that of CD45RA:958–973. Deletion of the acidic insert in CD45RO decreased the overall magnitude of the Ca²⁺ response, suggesting that the function of this isoform may be differentially regulated by CK2-dependent phosphorylation. In contrast, CD3-mediated activation of Erk1/2 and Jnk was affected to a similar extent in cells expressing CD45RA:958–973 and CD45RO:808–826 mutant molecules. Thus, it remains to be determined whether distinct CD45 isoforms are differentially regulated by CK2-dependent post-translational modification. Additional studies with the CD45RA isoform revealed that mutation of the CK2 phosphoacceptor sites within the acidic insert results in a similar shift in the kinetics of the Ca²⁺ response compared with the CD45RA mutant lacking the entire insert. This finding supports the conclusion that phosphorylation of specific serine residues within the insert may be directly involved in regulation of CD45 function as opposed to the association with CK2 per se. In support of this conclusion, the interaction between CK2 and the serine to alanine mutant of CD45 was only slightly decreased compared with that of wild-type CD45RA.

Previous studies have demonstrated that stable transfection of CD45-deficient H45.01 T cells with CD45RO in which the four serines within the DII acidic insert were mutated to alanine (Ser⁸¹⁵, Ser⁸¹⁸, Ser⁸¹⁹, and Ser⁸²³ to Ala) results in a sustained Ca²⁺ flux after TCR cross-linking, without affecting the magnitude of the response (42). In contrast, experiments performed in this study did not reveal a sustained elevation in the free intracellular concentration of Ca²⁺ in cells expressing CD45RO:808–826 compared with that in cells that express wild-type CD45RO (data not shown), whereas a significant decrease in the magnitude of the overall Ca²⁺ mobilization response was observed in response to CD3 cross-linking. Additionally, the CD3-mediated Ca²⁺ response was not sustained in Jurkat cells transfected with either CD45RA:958–973 or CD45RA:S965/968/969/973A compared with wild-type CD45RA (data not shown). Thus, both studies support the conclusion that the 19-aa insert is important for regulating CD45 function, although it is not clear whether CK2-dependent phosphorylation affects the ability of CD45 to regulate the initiation of the signaling response after TCR cross-linking and/or the resolution of the response. It is formally possible that experimental differences in the two studies, related to the cell lines and/or the activation stimuli used, could be responsible in part (42).

The mechanism by which CK2-dependent post-translational modification of CD45 regulates its function is unknown at present. Nevertheless, it is possible that phosphorylation of the insert alters the conformation of DII, which, in turn, regulates the formation of intramolecular bonds between DI and DII. Studies have shown that the catalytic activity of CD45 is negatively regulated by intermolecular dimerization (43, 44). This is thought to be due to the reciprocal insertion of a wedge located in the membrane-proximal region of one CD45 molecule into the substrate binding pocket in PTP DI of another. It has further been hypothesized that intermolecular dimerization and wedge insertion are regulated by the intramolecular association between DI and DII in a given CD45 molecule (20). Studies suggest that the formation of an intramolecular interaction between DI and DII prevents intermolecular dimerization, thus enhancing CD45 catalytic function (20, 45, 46). This prediction is supported by numerous studies demonstrating that the catalytic activity and/or substrate specificity of PTP DI are regulated by DII (18, 19, 20, 47). In this regard it is possible that phosphorylation of the acidic insert in DII may promote the formation of an intramolecular bond between DI and DII, resulting in increased CD45 activity. Another possible mechanism by which CK2-dependent phosphorylation of CD45 could regulate its function relates to the potential role that DII plays in substrate recruitment. Previous studies have shown that DII of CD45 appears to be required for optimal recruitment of selected substrates (21). Thus, it is possible that phosphorylation of the acidic insert that is located adjacent to the substrate binding pocket of DII might alter its affinity and/or specificity for substrates.

In summary, the results from this study support the conclusion that there is a direct physical interaction between CD45 and the CK2 holoenzyme, and that this interaction is important for post-translational modification of CD45 resulting in alteration of its catalytic activity and/or substrate specificity.

	Acknowledgments

 
We thank Dr. Matt Thomas for supplying the CD45 minigene constructs, Dr. Kerry Campbell for providing the p14 MET25 yeast vector, and Dr. David W. Litchfield for providing rabbit antiserum specific for CK2.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grant GM46524.

² S.F.G. and Y.W. contributed equally to this work and should be considered as co-first authors.

³ Address correspondence and reprint requests to Dr. Louis B. Justement, 378 Wallace Tumor Institute, Division of Developmental and Clinical Immunology, University of Alabama, Birmingham, AL 35294-3300. E-mail address: louis.justement{at}ccc.uab.edu

⁴ Abbreviations used in this paper: PTK, protein tyrosine kinase; AgR, Ag receptor; PTP, protein tyrosine phosphatase; BCR, B cell Ag receptor; DI, CD45 protein tyrosine phosphatase domain I; DII, CD45 protein tyrosine phosphatase domain II; CK2, casein kinase 2; BD, GAL4 binding domain; AD, GAL4 activation domain; Erk1/2, extracellular signal-regulated kinase 1/2; Jnk, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Received for publication November 10, 2000. Accepted for publication April 12, 2001.

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