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The Journal of Immunology, 1999, 162: 5278-5286.
Copyright © 1999 by The American Association of Immunologists

CD45 Regulates Tyrosine Phosphorylation of CD22 and Its Association with the Protein Tyrosine Phosphatase SHP-11

Susanna F. Greer and Louis B. Justement2

Department of Microbiology, Division of Developmental and Clinical Immunology, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transmembrane protein tyrosine phosphatase (PTP)3 CD45 plays a critical role in regulating lymphocyte development and activation (1). Studies have demonstrated that CD45 regulates the B cell Ag receptor (BCR) signaling threshold, thereby controlling both positive and negative selection (2, 3, 4, 5). Analysis of CD45-deficient cell lines and cells derived from gene-targeted mice has demonstrated that CD45 is involved in regulating reversible tyrosine phosphorylation and thus the intrinsic sensitivity of the BCR to a given antigenic challenge (4, 5, 6, 7, 8, 9). Additionally, CD45 has been shown to regulate signaling via MHC class II Ags expressed on the B cell in a manner analogous to that observed for the BCR (6). Thus, CD45 can regulate B cell development, activation, and differentiation by virtue of its ability to alter signaling thresholds associated with these receptors.

CD45 expression is required for optimal BCR- and class II-mediated activation of Src family protein tyrosine kinases (PTK), but not the PTK Syk (6, 8, 10). Indeed, BCR-mediated activation of Syk occurs in the absence of CD45, resulting in the phosphorylation and activation of phospholipase C{gamma} (PLC{gamma}), which hydrolyzes phosphatidylinositol 4,5-biphosphate to produce the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (6, 7). Despite the fact that IP3 appears to be produced in cells that lack CD45, the downstream Ca2+ mobilization response exhibits significant changes in both its amplitude and duration. Based on studies using the K46–17 µm{lambda} B cell lymphoma and derivative CD45-deficient mutants, receptor-proximal events associated with signal transduction through the BCR and class II are similarly affected by the loss of CD45 expression (6), although there are differences in the Ca2+ mobilization responses elicited by stimulating B cells through these receptors. Whereas signaling through the BCR elicits a relatively normal intracellular Ca2+ mobilization response in the absence of CD45, cross-linking of class II does not. In contrast, stimulation through either receptor fails to induce significant influx of Ca2+ from the extracellular space (6). Besides alterations in the Ca2+ response, Ras activation leading to signaling via the extracellular signal-related kinase 2 (ERK2) pathway is abrogated in cells that are CD45 deficient (3, 11). Clearly, then, CD45 plays an important role in regulating reversible tyrosine phosphorylation, which controls the production of second messengers (e.g., Ca2+) and the activation of distal signaling pathways (e.g., ERK2-dependent signaling responses) that regulate gene transcription (2, 12).

The specific molecular defect responsible for attenuation of the Ca2+ mobilization response in CD45-deficient B cells has not been clearly defined. Because CD45 is important for activation of Src family PTKs (6, 8, 10), it is possible that suboptimal Src PTK activation affects signal propagation, leading to decreased Ca2+ responses. However, several events that are distal to Src PTK activation, such as Syk activation, PLC{gamma} phosphorylation, and IP3 production, do not appear to be significantly affected in CD45-deficient cells (6, 7). It is formally possible that Src family PTK activation is important for the activation of other effector proteins that regulate Ca2+ mobilization. Alternatively, it is possible that the loss of CD45 leads to alterations in reversible tyrosine phosphorylation of downstream signaling proteins or coreceptors that, in turn, promote the recruitment/activation of inhibitory effector molecules that effectively attenuate the Ca2+ response. In support of this possibility, recent studies demonstrated that phosphorylation of the coreceptor CD22 is increased in cells that lack CD45 (6).

CD22 is a B cell-restricted transmembrane glycoprotein that functions as a coreceptor that regulates BCR-dependent signal transduction (13, 14, 15, 16). CD22 coreceptor function is regulated by Lyn-dependent tyrosine phosphorylation of its cytoplasmic domain, creating phosphotyrosine motifs that recruit multiple SH2 domain-containing effector proteins (17, 18, 19, 20). Recent studies suggest that CD22 can attenuate signal transduction via the BCR through its interaction with the PTP SHP-1 (21, 22). In particular, studies have demonstrated that CD22, presumably through its interaction with SHP-1, can attenuate Ca2+ mobilization in response to BCR cross-linking (21, 22). Thus, it was of interest to further explore the relationship between CD45 and CD22 and to determine whether aberrant tyrosine phosphorylation of CD22 in CD45-deficient cells is responsible for attenuation of Ca2+ mobilization following cross-linking of the BCR or MHC class II. In these studies a catalytically inactive mutant of SHP-1 was expressed in CD45-deficient B cells in an attempt to determine whether competitive inhibition of endogenous SHP-1 binding to CD22 restores normal signal transduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Biological reagents

The mAbs used in these studies were: B76 (rat IgG, anti-mouse µ heavy chain), JA12.5 (rat IgG2b, anti-mouse IgD), D3.137.5.7 (rat IgG2a, anti-mouse MHC class II, Iad,b haplotype), I3/2.3 (rat IgG2b, anti-mouse CD45), RG7/9.1 (mouse IgG2b, anti-rat {kappa} light chain), CY34 (mouse IgG1, anti-mouse CD22.2), and NIMR-6 (rat IgG1, anti-mouse CD22). mAbs were purified using protein G-Sepharose 4B Fast Flow (Amersham-Pharmacia Biotech, Piscataway, NJ) and were biotinylated using N-hydroxysuccinimidobiotin (Sigma, St. Louis, MO) as described previously (23). The Abs 4G10 (mouse IgG2b, anti-phosphotyrosine (PTyr), Upstate Biotechnology, Lake Placid, NY), PTP1C (mouse IgG1 anti-SHP1, Transduction Laboratories, Lexington, KY), and 9E10.2 (mouse IgG1, anti-c-Myc, American Type Culture Collection, Manassas, VA) were purchased for these studies. Polyclonal rabbit anti-Lyn was obtained from Dr. Joseph Bolen (DNAX, Palo Alto, CA). Polyclonal goat anti-mouse IgG coupled to horseradish peroxidase (HRPO) and goat anti-rabbit IgG coupled to HRPO were purchased from BioSource (Camarillo, CA). Streptavidin was purchased from Pierce (Rockford, IL). Dr. Ivan Stamenkovic (Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA) provided the soluble recombinant CD22 fusion protein used in these studies. The CD22ßRg fusion protein contains the first three Ig-like domains from CD22 fused to the heavy chain constant region of human IgG1 (24).

Cell lines

The B lymphoma cell line K46–17 µm{lambda} (K46) was provided by Dr. Michael Reth (Max-Planck Institut fur Immunbiologie, Freiburg, Germany). K46 cells were cultured 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. CD45-negative variants of the K46 line were isolated after chemical mutagenesis with methanesulfonic acid ethyl ester (EMS; Sigma) as described previously (6). Complement-mediated negative selection was used to remove CD45-positive cells, and FACS was used to enrich the resultant CD45-negative cell population. CD45-negative cells isolated by FACS were then cloned by limiting dilution, and several CD45-negative clones were examined. Two of these clones, 3S5 and 35S5, have been characterized as described previously (6), and the 35S5 cell line was used for these studies. The 35S5 cell line was transfected with either the empty pSFFV vector (vector control) or the pSFFV vector containing the cDNA encoding catalytically inactive SHP-1 (C453S mutant, SHP-1:C453S, provided by Dr. Matt Thomas, Department of Pathology, Washington University, St. Louis, MO). The SHP-1:C453S protein contains a Myc epitope tag on the carboxyl-terminal end to facilitate discrimination between endogenous and catalytically inactive forms of SHP-1. For transfection 1 x 107 K46 and 35S5 cells were resuspended in 500 µl of IMDM and transfected with 10 µg of cDNA using a Becton Dickinson electroporator (Mountain View, 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, Grand Island, NY). Lysates from drug-resistant transfectants were resolved by SDS-PAGE, and Western blotting was performed to detect expression of the c-Myc-tagged SHP-1:C453S mutant by sequentially probing membranes with anti-Myc Ab (9E10) followed by anti-SHP-1 Ab. Enhanced chemiluminescence (ECL) was used to visualize expression of endogenous and catalytically inactive SHP-1.

Immunoreceptor capping studies

To determine whether CD22 colocalizes in the plasma membrane with CD45, K46 cells (1 x 106) were washed in PBS with Ca2+ and were incubated in the presence of anti-CD45 mAb (biotinylated I3/2.3, 22 µg/ml) for 20 min on ice. The cells were washed in PBS and were incubated in PBS with Ca2+ in the presence of the secondary cross-linking reagent FITC-conjugated streptavidin (5 µg/ml; BioSource) for 30 min at 37°C. After stimulation cells were washed in Ca2+- and Mg2+-free PBS containing 2% sodium azide and 2% FBS and were incubated with anti-CD22 mAb (PE-conjugated NIMR-6, 10 µg/ml) for 20 min on ice. Control samples were prepared in which CD45 was not capped by performing all the incubation steps described above at 4°C. Subsequently, the cells were washed, and samples were prepared by cytospinning the cells onto glass slides. The cells were fixed by incubating the slides in ice-cold ethanol overnight. Fixed slides were washed and mounted in Fluoromount G (Southern Biotechnology Associates, Birmingham, AL) and viewed with a Leica/Leitz DMRB fluorescence microscope equipped with the appropriate filter cubes for two-color immunofluorescence (Chromatechnology, Brattleboro, VT).

Immunoprecipitation and immunoblotting

To analyze the effect that aggregation of CD45 has on CD22 phosphorylation, K46 cells (1 x 107) were incubated in medium alone or in the presence of anti-CD45 mAb (biotinylated I3/2.3, 10 µg/ml) for 15 min at room temperature. Cells were then washed twice in PBS and were resuspended in 1 ml of IMDM containing 5% FBS. The cells were rested at 37°C for 20 min, and streptavidin (10 µg/ml) was added, after which cells were incubated at 37°C for 1, 10, or 30 min. Control samples (NT) received neither anti-CD45 nor streptavidin but were incubated at 37°C for 30 min. Reactions were stopped by the addition of ice-cold PBS containing 1 mM sodium orthovanadate.

MHC class II- and BCR-mediated signaling events were analyzed as described previously (6). Briefly, to analyze MHC class II-mediated signaling, K46 cells, the CD45-negative variant 35S5, or the SHP-1:C453S transfectant of 35S5 (2.5 x 107 cells/sample) were incubated in medium alone (NT) or in the presence of biotinylated anti-class II mAb (D3.137.5.7, 10 µg/ml) for 15 min at room temperature. Cells were then washed in PBS, resuspended in IMDM, and rested at 37°C for 20 min, followed by incubation for 1, 10, or 30 min with streptavidin (10 µg/ml). Cells (2.5 x 107) stimulated through the BCR were harvested, rested at 37°C for 20 min, and incubated with medium alone (NT) or in the presence of mAb directed against the BCR (anti-IgM, B76, 10 µg/ml) for 1, 10, or 30 min.

After stimulation, cells were washed twice in ice-cold PBS and lysed in 0.5 ml of lysis buffer (25 mM HEPES, 150 mM NaCl (pH 7.8), 10 mM EDTA, 1 mM EGTA, 0.1 mM Na3VO4, and 1% Nonidet P-40). Cells were incubated for 1 h on ice, and the lysates were centrifuged at 12,000 x g for 15 min at 4°C. Detergent-soluble lysates were precleared by incubation with protein G-Sepharose (for SHP-1, c-Myc, and Lyn experiments) or RG7/9.1 bound to Sepharose 4B (for CD22 experiments) for 1 h at 4°C. Proteins to be analyzed were immunoprecipitated from precleared lysates either by the addition of soluble Abs (SHP-1, c-Myc, and Lyn) plus protein G-Sepharose or by the addition of mAb coupled directly to Sepharose 4B beads (CD22). Samples were incubated for 1 h at 4°C with rotation. Immune complex-coated beads were collected and washed four times with lysis buffer containing 0.2% Nonidet P-40. The beads were resuspended in 25 µl of SDS-PAGE sample reducing buffer, boiled for 4 min, and centrifuged. The proteins in 15 µl of supernatant from each sample were separated on 8% acrylamide gels by SDS-PAGE and were transferred to Hybond-ECL nitrocellulose membranes (Amersham-Pharmacia Biotech). The membranes were blocked with 10% nonfat dry milk in TBST for 1 h at room temperature and were washed five times with TBST. Next, the membranes were incubated with the appropriate primary Ab, washed five times with TBST, incubated with the appropriate HRPO-conjugated secondary Ab for 1 h at room temperature, washed five times with TBST, and visualized using ECL. In selected experiments, blots were stripped by incubation in stripping buffer (10 mM Tris (pH 2.3) and 150 mM NaCl) at 60°C for 1 h and then were washed extensively as described above. The membranes were then blocked with 3% blot qualified BSA (Promega, Madison, WI) in PBS, washed extensively, and incubated with anti-PTyr mAb (4G10) for 1 h. The membranes were washed and developed as described above using ECL.

Measurement of calcium mobilization

Studies were performed with K46 cells, the 35S5 clone, 35S5 vector control transfectants, and 35S5:SHP-1:C453S transfectants in which Ca2+ mobilization was assayed in response to BCR or MHC class II cross-linking as described previously (6). 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 (San Jose, CA) 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. Analysis of BCR- and MHC class II-mediated Ca2+ mobilization was performed as previously described (6). For class II Ca2+ response measurements, the cells were incubated with biotinylated anti-class II mAb (D3.137.5.7, 10 µg/ml, 15 min at room temperature) and washed, and an unstimulated baseline was established. Once a baseline measurement for intracellular free Ca2+ was recorded, streptavidin (10 µg/ml) was added, and the analysis was resumed. The BCR-mediated Ca2+ mobilization response was initiated by the addition of anti-µ (B76, 10 µg/ml) once a baseline was established. Measurement of the intracellular vs extracellular influx of Ca2+ was performed as previously described (6). Cells were resuspended in Ca2+- and Mg2+-free-PBS just before analysis, and a baseline was established. The cells were stimulated as described above with mAb directed against the BCR or MHC class II, and the resulting Ca2+ mobilization response was allowed to return to baseline. Once the baseline was resolved, CaCl2 was added to a final concentration of 4 mM, and measurement of extracellular Ca2+ influx was initiated.

Measurement of CD22-associated protein tyrosine phosphatase activity

Experiments to measure CD22-associated protein tyrosine phosphatase activity were performed using Tyrosine Phosphatase Assay Kit I from Upstate Biotechnology following the manufacturer’s protocol. The samples were prepared for assay by stimulating K46 cells, 35S5 CD45-negative variants, or 35S5:SHP-1:C453S transfectants (2 x 107/sample) with mAbs directed against the BCR as described previously. The cells were washed with PBS and lysed on ice in 0.5 ml of buffer containing 1% Nonidet P-40 without phosphatase inhibitors. The lysates were centrifuged and precleared, and CD22 was immunoprecipitated as described above. Immune complex-coated beads were washed once in wash buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Nonidet P-40), twice in WG buffer (50 mM HEPES (pH 7.6), 150 mM NaCl, and 0.1% Nonidet P-40), and twice in assay buffer (40 mM 2-(N-morpholino)ethanesulfonic acid and 1.6 mM DTT). The assays were performed as indicated in the protocol. An incubation time of 30 min at 30°C with phosphopeptide was determined to be optimal for measurement of CD22-associated PTP detection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Redistribution of CD45 leads to alterations in CD22 phosphorylation and effector protein recruitment

Previous studies have demonstrated that CD22, CD45, and the BCR physically interact with one another in the plasma membrane (25, 26, 27, 28), suggesting that a functional relationship may exist among these proteins. That this is true for the BCR and CD22 has been demonstrated by several laboratories in experiments showing that cross-linking of the BCR leads to increased tyrosine phosphorylation of CD22 (29, 30). Thus, it was of interest to determine whether perturbation of CD45 leads to alterations in the phosphorylation status of CD22 as well. Previous studies have been performed demonstrating that redistribution of CD45 within the plasma membrane of B cells can lead to alterations in the phosphorylation of other transmembrane proteins such as Ig{alpha} and Igß (31). Therefore, experiments were performed to determine whether Ab-mediated cross-linking and redistribution of CD45 affect CD22 phosphorylation and recruitment of intracellular effector proteins. First, experiments were performed to determine whether CD22 and CD45 colocalize in the membrane when CD45 is cross-linked and capped. In unstimulated B cells, both FITC-labeled CD45 and PE-labeled CD22 exhibit a uniform distribution throughout the plasma membrane (Fig. 1GoA). By comparison, when cells were incubated with biotinylated anti-CD45 followed by FITC-streptavidin, there was a marked capping of CD45, with little or no concurrent redistribution of CD22 in the membrane (Fig. 1GoB).



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  		FIGURE 1. CD22 and CD45 do not colocalize in the plasma membrane in response to CD45 cross-linking. Cells (1 x 106) were washed in PBS with Ca2+ and were incubated in the presence of anti-CD45 mAb (biotinylated I3/2.3, 22 µg/ml) for 20 min on ice. These cells were washed in PBS with Ca2+ and were incubated in PBS with Ca2+ alone (A) or in the presence of a secondary cross-linking reagent (B; FITC-conjugated streptavidin, 5 µg/ml) for 30 min at 37°C. After stimulation cells were washed in PBS without Ca2+ and Mg2+ containing 2% sodium azide and 2% FBS and were incubated with anti-CD22 mAb (PE-conjugated NIMR-6) for 20 min on ice. Slides were prepared by cytospinning the cells onto glass slides, after which they were incubated in ice-cold ethanol overnight. Fixed slides were washed and mounted in Fluoromount G and viewed with a Leica/Leitz DMRB fluorescence microscope equipped with appropriate filter cubes.

 
Next, experiments were performed to determine whether redistribution of CD45 in the plasma membrane affects CD22 phosphorylation. CD45 was cross-linked with anti-CD45 mAb (biotinylated I3/2.3) plus streptavidin, and CD22 was immunoprecipitated from cell lysates. Western blotting was performed to measure tyrosine phosphorylation of CD22, revealing an increase in its tyrosine phosphorylation in conjunction with cross-linking of CD45 (Fig. 2Go). Because CD22 has been shown to play a role in negatively regulating BCR signal transduction through its tyrosine phosphorylation-dependent interaction with the PTP SHP-1 (22), the blot was stripped and reprobed for SHP-1. A concomitant increase in binding of SHP-1 to tyrosine-phosphorylated CD22 was seen in response to CD45 cross-linking (Fig. 2Go). These data suggest that the alterations seen in CD22 phosphorylation may be due to physical sequestration of CD45 in the plasma membrane or inhibition of its catalytic function resulting from mAb-mediated multimerization. In either case, it appears that the basal phosphorylation status of CD22 is regulated by CD45 either directly or indirectly through other PTKs or PTPs that are substrates for CD45.



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  		FIGURE 2. Redistribution of CD45 in the membrane leads to tyrosine phosphorylation of CD22 and recruitment of SHP-1. Cells (1 x 107) were incubated in medium alone or in the presence of anti-CD45 mAb (biotinylated I3/2.3, 10 µg/ml) for 15 min at room temperature. Cells were then washed twice in PBS and were resuspended in 1 ml of IMDM containing 5% FBS. The cells were rested at 37°C for 20 min, and streptavidin (10 µg/ml) was added, after which cells were incubated at 37°C for varied periods of time. Control samples (NT) received neither anti-CD45 nor streptavidin, but were incubated at 37°C for 30 min. Reactions were stopped by the addition of ice-cold PBS containing 1% Nonidet P-40, and CD22 was immunoprecipitated with mAb NIMR-6 coupled to Sepharose 4B. Proteins were separated by 8% SDS-PAGE and were transferred to nitrocellulose. Tyrosine phosphorylation of CD22 was visualized by probing the membrane with anti-PTyr mAb coupled to HRPO (4G10) followed by development of the blot with ECL (left panel). The membrane was stripped and reprobed with anti-SHP-1 mAb followed by goat anti-mouse IgG coupled to HRPO and again visualized using ECL (right panel).

 
CD22 is hyperphosphorylated in the absence of CD45, resulting in enhanced recruitment of SHP-1

To further examine the potential role of CD45 in regulating tyrosine phosphorylation of CD22, experiments were performed using CD45-deficient variants of the K46 cell line. These CD45-deficient cells were generated by chemical mutagenesis and have been characterized previously (6). K46 parental cells and the CD45-negative 35S5 mutant were stimulated through the BCR with anti-IgM mAb (B76), CD22 was immunoprecipitated, and tyrosine phosphorylation of CD22 was examined by Western blotting. As depicted in Fig. 3Go, CD22 was inducibly phosphorylated in response to anti-IgM regardless of whether CD45 was expressed. However, consistent differences in CD22 phosphorylation were observed in the absence of CD45, including an elevation in the basal level of tyrosine phosphorylation and pronounced hyperphosphorylation in response to BCR cross-linking (Fig. 3Go). When the membrane was stripped and reprobed with Ab directed against SHP-1, it was noted that the amount of SHP-1 associated with CD22 was significantly increased in the CD45-negative cells (3- to 5-fold). These findings suggest that loss of CD45 expression affects both the basal and inducible phosphorylation status of CD22, which, in turn, affects the recruitment of SHP-1 to the membrane. Additional studies were performed to determine whether the reciprocal experiment in which SHP-1 was immunoprecipitated after BCR cross-linking would yield comparable results. SHP-1 was isolated from K46 and 35S5 CD45-negative cells, and Western blotting was used to measure its association with phosphotyrosine-containing proteins. As depicted in Fig. 4Go, SHP-1 isolated from 35S5 cells exhibited an 8- to 10-fold increase in its binding to a tyrosine-phosphorylated protein migrating at 140 kDa compared with SHP-1 isolated from K46 parental cells. When the membrane was stripped and reprobed with Ab directed against SHP-1, it was noted that the amount of SHP-1 in each lane was comparable (Fig. 4Go).



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  		FIGURE 3. BCR cross-linking induces hyperphosphorylation of CD22 on tyrosine and increased recruitment of SHP-1 in CD45-deficient B cells. Experiments were performed with K46 cells and the CD45-negative clone 35S5 (1 x 107 cells/sample) in which the cells were incubated for varied periods of time (1–30 min) in medium alone (NT) or in the presence of mAbs directed against the BCR (anti-IgM, B76, 10 µg/ml). At the appropriate time, cells were resuspended in ice-cold PBS, washed, and lysed in buffer containing 1% Nonidet P-40. Lysates were precleared, and CD22 was immunoprecipitated with the mAb NIMR-6 coupled to Sepharose 4B. Proteins were separated by 8% SDS-PAGE and were transferred to nitrocellulose. Tyrosine phosphorylation of CD22 was visualized by probing the membrane with anti-PTyr mAb coupled to HRPO (4G10) followed by development of the blot with ECL (top panel). The membrane was stripped and reprobed with anti-SHP-1 mAb followed by goat anti-mouse IgG coupled to HRPO, and SHP-1 was visualized using ECL (lower panels).

 


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  		FIGURE 4. BCR cross-linking leads to enhanced association between SHP-1 and tyrosine-phosphorylated proteins in CD45-deficient B cells. Cells were stimulated and lysed as described above (see Fig. 5Go). SHP-1 was immunoprecipitated from precleared lysates using a mouse anti-SHP-1 mAb and protein G-Sepharose. Immune complex material was washed extensively and separated by 8% SDS-PAGE. Resolved proteins were transferred to nitrocellulose, and SHP-1-associated tyrosine phosphoproteins were visualized by probing the membrane with 4G10 coupled to HRPO (top panel). The membrane was stripped and reprobed with anti-SHP-1 mAb as described above to demonstrate equal recovery and loading of SHP-1 (lower panel).

 
Studies have shown that CD45 regulates signaling via MHC class II molecules and the BCR in an analogous manner (6). Therefore, experiments were performed to evaluate whether cross-linking of class II leads to hyperphosphorylation of CD22 in the absence of CD45. Cross-linking of MHC class II was observed to induce phosphorylation of CD22 regardless of whether CD45 was present (Fig. 5Go). As seen previously, the CD45-negative cells exhibited both basal and inducible hyperphosphorylation of CD22 compared with CD45-positive K46 cells. When the membrane was stripped and reprobed to detect SHP-1, the amount of SHP-1 bound to CD22 was several times greater (>10-fold) in the CD45-negative cells than in the K46 parental cells. Thus, CD45 plays a comparable role in regulating CD22 phosphorylation via BCR- and MHC class II-dependent signaling pathways in the B cell.



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  		FIGURE 5. Cross-linking of MHC class II induces hyperphosphorylation of CD22 on tyrosine and increased recruitment of SHP-1 in CD45-deficient B cells. Experiments were performed with K46 cells and the CD45-negative clone 35S5 (1 x 107 cells/sample) in which cells were incubated in medium alone (NT) or in the presence of biotinylated anti-class II mAb (D3.137.5.7, 10 µg/ml) for 15 min at room temperature. Cells were then washed in PBS, resuspended in IMDM with 5% FCS, and incubated for various times with streptavidin at a final concentration of 10 µg/ml. At the appropriate time points, cells were resuspended in ice-cold PBS, washed, and lysed in buffer with 1% Nonidet P-40, and CD22 was immunoprecipitated as previously indicated. Tyrosine phosphorylation of CD22 (top panel) and SHP-1 recruitment (lower panel) were visualized as described previously.

 
Lyn associates with CD22 in CD45-deficient cells

Expression of the Src family kinase Lyn has recently been shown to be essential for inducible tyrosine phosphorylation of CD22 in response to BCR cross-linking (17, 18, 19, 20). Moreover, previous studies have demonstrated that CD45 expression is required for activation of the Src family kinases, including Lyn (6, 8, 10). We have shown that in the CD45-deficient 35S5 cell line, activation of Lyn is attenuated in response to BCR or class II cross-linking (6). Because of the functional relationship among CD45, Lyn, and CD22, the finding that BCR or class II cross-linking leads to hyperphosphorylation of CD22 in the absence of CD45 expression was not expected. It was therefore of interest to further examine the relationship between CD22 and Lyn in the 35S5 cell line. It has been shown that Lyn is recruited to CD22 in response to stimulation through the BCR (32, 33). The nature of the physical interaction between Lyn and CD22 has yet to be fully elucidated, although it may be mediated at least in part by a phosphotyrosine-independent mechanism (33).

Experiments were conducted in which K46 and 35S5 cells were stimulated with anti-IgM (B76) mAb followed by immunoprecipitation of CD22 from detergent-solubilized lysates. Western blot analysis to measure tyrosine phosphorylation revealed inducible phosphorylation of CD22 with hyperphosphorylation in the CD45-negative cells as described above (data not shown). When the membrane was stripped and reprobed to detect Lyn (Fig. 6Go), it was noted that the amount of Lyn associated with CD22 isolated from the 35S5 cell line, compared with that from K46 parental cells, was elevated in unstimulated cells and at early time points (1–10 min) after treatment with anti-Ig. Indeed, whereas the interaction between Lyn and CD22 appeared to be inducible in K46 cells, it was essentially constitutive in cells lacking CD45. Similar results were obtained when the CD45-deficient 3S5 cell line was examined (data not shown). These findings suggest that even though the overall activation of Lyn may be attenuated in the absence of CD45, its association with CD22 in the 35S5 cells is unaffected, thereby providing a potential explanation for the increased basal and inducible levels of CD22 tyrosine phosphorylation.



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  		FIGURE 6. CD45 expression is not required for the physical association between CD22 and the PTK Lyn. K46 and 35S5 (1 x 107/sample) cells were incubated in medium alone (NT) or in the presence of anti-IgM mAb (B76, 10 µg/ml) for the amount of time indicated. mAb-mediated stimulation of B cells was stopped by the addition of ice-cold PBS. The cells were washed and resuspended in buffer containing 1% Nonidet P-40 for 1 h on ice. The lysates were precleared, and CD22 was immunoprecipitated using the mAb NIMR-6 coupled to Sepharose 4B. Immune complexes were resolved by SDS-PAGE on 10% acrylamide gels, and the proteins were transferred to nitrocellulose. The association of Lyn was detected using a polyclonal rabbit anti-mouse Lyn Ab followed by the addition of a secondary goat anti-rabbit Ig Ab coupled to HRPO. The Lyn band was visualized using ECL.

 
Expression of catalytically inactive SHP-1 restores the intracellular Ca2+ mobilization response in CD45-deficient cells

Previous studies have shown that the loss of CD45 expression has a significant effect on the BCR-mediated Ca2+ mobilization response in B cells (6, 34). The 35S5 CD45-negative cell line exhibits little or no extracellular influx of Ca2+ in response to BCR cross-linking, whereas the mobilization of Ca2+ from intracellular stores is relatively unaffected (6). In contrast, a more pronounced effect on class II signaling is observed in which both components of the Ca2+ mobilization response are significantly attenuated (6). Because CD22 was observed to be hyperphosphorylated in the 35S5 CD45-negative cell line, leading to enhanced recruitment of SHP-1, experiments were performed to determine whether the aberrant recruitment of SHP-1 is responsible for attenuation of signal transduction via the BCR and MHC class II activation complexes.

Catalytically inactive SHP-1 with a C453S mutation (SHP-1:C453S) was transfected into 35S5 cells. The C453S mutation in SHP-1 inhibits its ability to catalyze the dephosphorylation of substrates, but not its ability to bind to them (35). To identify transfectants that expressed the catalytically inactive SHP-1:C453S mutant, lysates from 35S5 cells transfected with vector alone and with vector containing the SHP-1:C453S cDNA were incubated with anti-Myc mAb, and the immune complex material was subjected to immunoblot analysis to determine the levels of Myc-tagged SHP-1:C453S expressed in the cells. As depicted in Fig. 7GoA, neither the 35S5 nor the 35S5 vector control cells expressed Myc-tagged SHP-1:C453S, whereas 35S5 cells transfected with the SHP-1:C453S cDNA expressed significant amounts of the catalytically inactive PTP. To further analyze 35S5:SHP-1:C453S transfectants, experiments were performed to assay binding of Myc-tagged SHP-1:C453S to CD22. As depicted in Fig. 7GoB, when the 35S5:SHP-1:C453S cells were stimulated through the BCR, and CD22 was immunoprecipitated, immunoblotting for Myc-tagged SHP-1:C453S demonstrated that the catalytically inactive PTP binds to CD22. Additionally, the association between Myc-tagged SHP-1:C453S and CD22 appeared to be inducible, although it should be noted that significant binding of SHP-1:C453S to CD22 could be detected in unstimulated cells.



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  		FIGURE 7. A catalytically inactive SHP-1 mutant expressed in CD45-deficient B cells associates with CD22 in response to BCR cross-linking. A, 35S5 cells were transfected with empty vector (35S5: VC) or with vector containing the cDNA encoding a Myc-tagged catalytically inactive mutant of SHP-1 with a C453S mutation (35S5: SHP-1: C453S). Transfectants were selected in the presence of G418 and were examined by Western blotting to detect the presence of Myc-tagged SHP-1. Lysates were prepared from 35S5 cells and transfectant populations (1 x 107 cells/sample). Next, Myc-tagged proteins were immunoprecipitated from each lysate using 9E10 mAb (10 µg/ml) and protein G-Sepharose. The immune complexes were resolved by SDS-PAGE, and the proteins were transferred to nitrocellulose. The presence of Myc-tagged SHP-1 was detected by probing the membranes with anti-Myc mAb (9E10) followed by the addition of a secondary rabbit anti-mouse Ig Ab coupled to HRPO. ECL was used to visualize Myc-tagged SHP-1. B, The association of catalytically inactive SHP-1 with CD22 was examined using Western blotting. 35S5: SHP-1: C453S cells (1 x 107/sample) were incubated in medium alone (NT) or in the presence of anti-IgM mAb (B76, 10 µg/ml) for 1–10 min. The cells were washed in ice-cold PBS and were lysed in buffer containing 1% Nonidet P-40. Lysates were precleared, and CD22 was immunoprecipitated with the anti-CD22 mAb NIMR-6 coupled to Sepharose 4B beads. The immune complexes were resolved by SDS-PAGE and were transferred to nitrocellulose. Tyrosine phosphorylation of CD22 was detected using the mAb 4G10 coupled to HRPO. The membrane was stripped and reprobed to detect the presence of Myc-tagged SHP-1 as described above.

 
Subsequent experiments were performed to measure the relative levels of PTP activity associated with CD22 in K46, 35S5, and 35S5:SHP-1:C453S cell lines. CD22 was immunoprecipitated from unstimulated and anti-Ig-treated cell lines, and the PTP activity associated with it was assayed as described in Materials and Methods. As expected, CD22 isolated from the unstimulated 35S5 cell line was associated with a 2- to 3-fold increase in the amount of PTP activity compared with that in K46 parental cells. After stimulation with anti-Ig, the amount of PTP activity associated with CD22 isolated from 35S5 cells was 10-fold greater. In contrast, the amount of PTP activity associated with CD22 isolated from anti-Ig-treated 35S5:SHP-1:C453S transfectants was 1.5- to 2-fold above that seen for the K46 parental cells. These data indicated that catalytically inactive SHP-1:C453S was effectively competing with endogenous SHP-1 for binding sites on CD22, thereby decreasing the total PTP activity associated with CD22 in the CD45-negative cells. These experiments confirmed that the 35S5:SHP-1:C453S transfectant cell line was an appropriate system in which to analyze whether aberrant recruitment of SHP-1 to CD22 results in attenuation of signal transduction.

Loss of CD45 expression has a more profound effect on the Ca2+ mobilization response associated with cross-linking of MHC class II. Therefore, studies were performed to determine whether expression of the SHP-1:C453S mutant restores signaling via MHC class II in CD45-deficient B cells. Analysis of Ca2+ mobilization in response to class II cross-linking revealed that the response is almost completely abrogated in the absence of CD45 (Fig. 8GoA, compare K46 vs 35S5 cells). Expression of the SHP-1:C453S mutant, however, partially restores the MHC class II-mediated Ca2+ response in the absence of CD45 (Fig. 8GoA, compare K46 with 35S5:SHP-1:C453S). As can be seen, the response in the SHP-1:C453S transfectants exhibits a transient phenotype characteristic of Ca2+ mobilization from intracellular stores without subsequent influx of Ca2+ from the extracellular space. This was confirmed by performing the assay in the presence of EGTA (data not shown). Because signaling through the BCR leads to mobilization of Ca2+ from intracellular stores even in the absence of CD45, it was of interest to determine whether the SHP-1:C453S mutant could restore the extracellular influx component of the BCR response in CD45-deficient cells. As depicted in Fig. 8GoB, expression of catalytically inactive SHP-1:C453S does not significantly alter the BCR-mediated Ca2+ mobilization response in cells lacking CD45. These findings suggest that aberrant recruitment and activation of SHP-1 in CD45-deficient cells may be involved in regulating mobilization of Ca2+ from intracellular stores in response to class II cross-linking. However, it does not appear that increased activation of SHP-1 is responsible for the lack of Ca2+ influx from the extracellular space in cells that are CD45-deficient regardless of whether they are stimulated through the BCR or class II.



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  		FIGURE 8. Expression of catalytically inactive SHP-1 in the CD45-deficient 35S5 B cell line restores mobilization of Ca2+ from intracellular stores in response to class II cross-linking. K46 parental cells, 35S5, 35S5: vector control (VC), and 35S5: SHP-1: C453S transfectants (1 x 106 cells/sample) were loaded with indo-1 as described in Materials and Methods. Experiments were then performed in which Ca2+ mobilization was assayed in response to stimulation through MHC class II (A; 10 µg/ml biotinylated D3.137 and 10 µg/ml streptavidin) or the BCR (B; B76, 10 µg/ml). For the analysis of class II-mediated Ca2+ mobilization, cells were incubated with anti-class II mAb for 10 min at room temperature. The cells were washed, and the Ca2+ baseline was established. The analysis was stopped, and streptavidin was added, after which the analysis was resumed immediately. The response to BCR cross-linking was measured by establishing an unstimulated baseline after which the analysis was stopped, and anti-Ig mAb was added. The analysis was reinitiated immediately after the addition of anti-Ig mAb. The Ca2+ ionophore ionomycin was used to ensure that cells were loaded equivalently with indo-1 (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have presented findings that support the hypothesis that CD45 and CD22 exist in the B cell plasma membrane as part of a multimolecular complex (24, 25, 26, 27, 28, 32, 35, 36). The results presented in the current study further indicate that a functional relationship exists between CD45 and CD22 based on the observation that CD45 is involved in regulating the tyrosine phosphorylation status of CD22. This conclusion is based on two experimental approaches. First, it was shown that mAb-mediated redistribution of CD45 in the plasma membrane is associated with enhanced tyrosine phosphorylation of CD22. Whether cross-linking of CD45 simply restricts its ability to physically engage substrates in the cell (e.g., CD22), results in inhibition of its catalytic function due to dimerization, or both is not known. Previous studies have suggested that CD45 cross-linking and physical sequestration in the membrane lead to enhanced tyrosine phosphorylation of putative substrates without affecting CD45 catalytic activity (31). Alternatively, studies have shown that ligand binding to an EGF:CD45 chimeric protein leads to dimerization of chimeric CD45 and inhibition of its catalytic function due to the reciprocal insertion of a wedge domain from one polypeptide into the PTP active site of the other, thereby preventing substrate binding (37, 38). Clearly, the processes of physical sequestration and inhibition of catalytic activity are not mutually exclusive and may both be involved in regulating CD45 function in vivo.

A second approach used in the current study to demonstrate that CD45 is involved in regulating CD22 phosphorylation was to compare the basal and inducible tyrosine phosphorylation of CD22 isolated from CD45-positive (K46) and CD45-deficient (35S5) B cell lines. The results obtained demonstrate that both basal and inducible phosphorylation of CD22 are increased in cells that lack CD45. These findings corroborate the results from the CD45 cross-linking experiments. Nevertheless, neither approach conclusively proves that CD22 is a direct substrate for CD45. It is equally likely that cross-linking of CD45 or elimination of its expression alters the activity of other PTPs or PTKs and that perturbation of the normal homeostasis of reversible tyrosine phosphorylation indirectly leads to increased phosphorylation of CD22.

Previous studies have demonstrated that the PTK Lyn plays a critical role in mediating inducible tyrosine phosphorylation of CD22 (17, 18, 19, 20). It was therefore surprising to find that CD22 is hyperphosphorylated in CD45-deficient B cells, indicating that Lyn is able to function in the absence of CD45 with respect to CD22 phosphorylation. Because studies have demonstrated that Lyn binds to CD22 via an undefined mechanism, and that Lyn can be recruited to CD22 in an inducible manner in response to BCR cross-linking (32, 33), it was hypothesized that this interaction is important for Lyn-dependent phosphorylation of CD22. Indeed, Lyn was observed to coprecipitate with CD22 regardless of CD45 expression. Moreover, Lyn appeared to be constitutively associated with CD22, and the amount of Lyn bound to CD22 was elevated in the absence of CD45 compared with that in K46 parental cells. The mechanism responsible for enhanced recruitment of Lyn in the absence of CD45 expression is not known at present. Although previous studies indicate that the association may not be entirely dependent on tyrosine phosphorylation of CD22 (33), suggesting that Lyn may interact with CD22 via residues in its unique amino-terminal region. This could facilitate Lyn-dependent basal phosphorylation of CD22, leading to hyperphosphorylation in the absence of CD45 and increased recruitment of Lyn via a phosphotyrosine-dependent binding process.

Binding of Lyn to CD22 via a phosphotyrosine:SH2 domain interaction could promote Lyn activation even though CD45 is not present. It is formally possible that tyrosine phosphorylation of CD22 generates phosphotyrosine motifs in its cytoplasmic tail that bind to the SH2 domain of Lyn with high affinity such that they effectively compete with the carboxyl-terminal phosphotyrosine residue in Lyn. Displacement of the Lyn carboxyl-terminal inhibitory phosphotyrosine residue by binding of its SH2 domain to one or more phosphotyrosine motifs in the cytoplasmic tail of CD22 would be predicted to derepress the enzymatic activity of Lyn. Thus, even though the overall level of Lyn activity may be decreased in CD45-deficient cells, the catalytic activity of Lyn associated with CD22 would be unaffected. An alternative explanation is that Lyn could act as an intermediate linker protein that facilitates the recruitment of another PTK that actually phosphorylates CD22. In this scenario Lyn activation would not be essential for CD22 phosphorylation, whereas its physical association with CD22 would. It is interesting to note that none of the studies examining Lyn knockout mice has definitively proven that Lyn phosphorylates CD22. These studies have correlated Lyn expression with CD22 phosphorylation, which proves that Lyn has to be present in the cell, although they have not demonstrated that Lyn itself phosphorylates CD22.

Hyperphosphorylation of CD22 in CD45-deficient B cells was observed to correlate with enhanced recruitment of the PTP SHP-1. Because the 35S5 CD45-deficient B cell line has been shown to exhibit decreased Ca2+ mobilization in response to BCR and MHC class II cross-linking (6), it was of interest to determine whether this was due to aberrant recruitment of SHP-1 to CD22. To address this question a catalytically inactive form of SHP-1 with a C453S point mutation was expressed in the 35S5 cell line to compete with endogenous wild-type SHP-1 for binding to phosphorylated CD22. Indeed, it was observed that the SHP-1 C453S mutant was able to compete with endogenous SHP-1 for binding to CD22, thereby restoring the associated PTP activity to a level comparable to that observed in CD45-positive cells. Nevertheless, catalytically inactive SHP-1 did not fully restore the Ca2+ mobilization response elicited by cross-linking of either the BCR or MHC class II. Although intracellular mobilization of Ca2+ was potentiated by expression of the C453S SHP-1 mutant, particularly in response to class II cross-linking, the influx of Ca2+ from the extracellular space was not restored regardless of the stimulus.

Previous studies have demonstrated that crossing CD45 knockout mice with the motheaten viable strain, which expresses a mutant form of SHP1 that is catalytically defective, corrects developmental abnormalities associated with the loss of either PTP alone (39). Moreover, functional elimination of both PTPs was observed to restore BCR-mediated signal transduction processes such as PTK and MAP kinase activation. These data suggest that CD45 and SHP-1 are functionally interrelated and that they act in a coordinate manner to regulate BCR signaling, leading to activation and maturation. The results presented in the current study support the concept that CD45 and SHP-1 functionally counterbalance one another to regulate specific signal transduction processes. However, it is unlikely that these PTPs have completely overlapping regulatory functions because expression of catalytically inactive SHP-1 fails to restore long term, receptor-mediated influx of Ca2+ from the extracellular space.

Based on the results obtained, it is likely that CD45 regulates distinct signaling components that control Ca2+ influx. The Ca2+ response seen in the CD45-deficient 35S5 cells is similar to that observed when the Fc{gamma}RIIb receptor is coligated with the BCR (40, 41). This raises the possibility that loss of CD45 expression leads to dysregulation of SH2-containing inositol 5'-phosphatase (SHIP) function that could, in turn, affect phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) production and BTK activation (42). Attenuation of PtdIns-3,4,5-P3 production or BTK activation could have a direct effect on the Ca2+ influx response (43, 44). Inhibition of PtdIns-3,4,5-P3 production or BTK activation could also indirectly affect capacitative entry of extracellular Ca2+ through a decrease in the PLC{gamma}-dependent production of IP3 (45). Alternatively, dysregulation of Src PTK activation in CD45-deficient cells could attenuate activation of Btk and PLC{gamma} (45, 46). Finally, it has been proposed that calcium channel function may be regulated by reversible tyrosine phosphorylation (47, 48). Therefore, it is formally possible that CD45 could be involved in regulating the function of calcium release-activated channels in the plasma membrane, either directly by dephosphorylating the channels or indirectly through regulation of PTK/PTP activity in the B cell. Further elucidation of the specific nature of the defect in Ca2+ mobilization is of interest because it will provide important information concerning the mechanism(s) by which CD45 regulates transcription factor activation and thus the threshold of signaling through the BCR.


   Acknowledgments
 
We thank Dr. M. L. Thomas for providing the cDNA encoding the Myc-tagged C453S SHP-1 mutant, Dr. I. Stamenkovic for supplying the CD22ßRg fusion protein, Dr. J. Bolen for the anti-Lyn polyclonal Ab, and Dr. M. Reth for allowing us to use the K46–17 µm{lambda} cell line.


   Footnotes
 
1 This work was supported by Grants GM46524 and AI36401 from the National Institutes of Health (to L.B.J.) and by Training Grant AI07051 from the National Institutes of Health (to S.F.G.). Back

2 Address correspondence and reprint requests to Dr. Louis B. Justement, Department of Microbiology, Division of Developmental and Clinical Immunology, 378 Wallace Tumor Institute, 1824 6th Avenue South, Birmingham, AL 35294-3300. E-mail address: Back

3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; BCR, B cell Ag receptor; PTK, protein tyrosine kinase; PLC{gamma}, phospholipase C{gamma}; IP3, inositol 1,4,5-trisphosphate; PTyr, phosphotyrosine; HRPO, horseradish peroxidase; IMDM, Iscove’s modified Dulbecco’s medium; EMS, methane sulfonic acid ethyl ester; ECL, enhanced chemiluminescence; PE, phycoerythrin; NT, nontreated control samples; SHIP, SH2-containing inositol 5'-phosphatase; PtdIns-3,4,5-P3, phosphatidylinositol-3,4,5-trisphosphate. Back

Received for publication September 15, 1998. Accepted for publication February 10, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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Siglecs--the major subfamily of I-type lectins
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S.-Y. Lin, S. Raval, Z. Zhang, M. Deverill, K. A. Siminovitch, D. R. Branch, and B. Haimovich
The Protein-tyrosine Phosphatase SHP-1 Regulates the Phosphorylation of {alpha}-Actinin
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S. Li, D. T. Ross, M. E. Kadin, P. O. Brown, and M. A. Wasik
Comparative Genome-Scale Analysis of Gene Expression Profiles in T Cell Lymphoma Cells during Malignant Progression Using a Complementary DNA Microarray
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M. Fouillit, R. Joubert-Caron, F. Poirier, P. Bourin, E. Monostori, M. Levi-Strauss, M. Raphael, D. Bladier, and M. Caron
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