Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center This information is current as of September 28, 2021. Zeljka Korade Mirnics, Eva Caudell, YanHua Gao, Kazuhiko Kuwahara, Nobuo Sakaguchi, Tomohiro Kurosaki, Joan Burnside, Károly Mirnics and Seth J. Corey J Immunol 2004; 172:4133-4141; ; doi: 10.4049/jimmunol.172.7.4133 Downloaded from http://www.jimmunol.org/content/172/7/4133

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center1

Zeljka Korade Mirnics,2* Eva Caudell,† YanHua Gao,* Kazuhiko Kuwahara,‡§ Nobuo Sakaguchi,‡ Tomohiro Kurosaki,¶ Joan Burnside,ʈ Ka«roly Mirnics,2# and Seth J. Corey2*†

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, Downloaded from immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously as- sociated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-␬B, elongation factor 1␣, CD79b, octamer binding factor 1, Ig H chain, stathmin, and ␥-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal http://www.jimmunol.org/ centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn3PU.13GANP. The Journal of Immunology, 2004, 172: 4133Ð4141.

timulation of the B cell receptor (BCR)3 activates signal- Tec, and Csk families (3–6). BCR evoked tyrosine phosphoryla- ing cascades that lead to proliferation, differentiation, and tion of Lyn, and Syk triggers a biochemical cascade in which a S effector functions (1–3). BCR activation recruits members variety of cellular proteins become tyrosine phosphorylated (3, 7). by guest on September 28, 2021 of nonreceptor protein tyrosine kinases (PTKs) of the Src, Syk, Although native B cell express several Src family PTKs (Lyn and Blk), the chicken B cell DT40 expresses only Lyn (8). Src PTKs are believed to have overlapping expression and redundant *Department of Pediatrics, University of Pittsburgh, School of Medicine, Children’s function (9). However, because Lyn is the only Src PTK family Hospital of Pittsburgh, Pittsburgh, PA 15213; †Division of Pediatrics, University of Texas-M. D. Anderson Cancer Center, Houston, TX 77030; ‡Department of Immu- member expressed in DT40 cells, this cell line provides a unique nology, Graduate School of Medical Sciences, Kumamoto University School of Med- experimental system to study Lyn’s role in B cell signaling. A icine, Honjo, Kumamoto, Japan; §PRESTO, Japan Science and Technology Corpo- unique feature of DT40 cells is their ability to undergo efficient ration, Kawaguchi, Japan; ¶Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan; ʈDelaware Biotechnology homologous recombination, so that more than several dozen genes Institute, University of Delaware, Newark, DE 19711; and #Departments of Psychi- have been inactivated and resulting lines intensively studied (10). atry and Neurobiology, PittArray Microarray Core, University of Pittsburgh, Pitts- burgh, PA 15261 Lyn-deficient DT40 cells show a defective BCR cross-linking re- sponse, manifested by slow calcium mobilization and profoundly Received for publication July 7, 2003. Accepted for publication February 2, 2004. reduced phosphorylation/activation of Syk (11). Lyn-deficient The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cells also show increased sensitivity to apoptosis; however, precise with 18 U.S.C. Section 1734 solely to indicate this fact. molecular deficiencies have not yet been associated with the Ϫ/Ϫ 1 Z.K.M. was supported by the Caligiuri Fund of the Pittsburgh Foundation, the Lupus Lyn phenotype. Foundation, the Bear Necessities Pediatric Cancer Foundation, and Children’s Hos- Lyn-deficient mice have a dramatic phenotype that resembles pital of Pittsburgh Research Advisory Committee. J.B. is supported by U.S. Depart- ment of Agriculture Grant NRICGP 98-35205-6640. K.M. is supported by National systemic lupus erythematosus in humans (12, 13). The B cells of Institutes of Health Grant R21MH/NS62760-01. S.J.C. is supported by National In- these mice show reduced response to BCR cross-linking or LPS stitutes of Health K02HL03794 and R29CA74422, and American Cancer Society Grant DHP-135. activation (14). With age, these mice develop high serum IgM levels, lymphadenopathy, and splenomegaly (15). In contrast, de- 2 Address correspondence and reprint requests to Dr. Seth J. Corey, Division of Pe- diatrics, University of Texas-M. D. Anderson Cancer Center, 1515 Holcombe Bou- ficiency of other Src PTK family members expressed predomi- levard, Houston, TX 77030; or Dr. Zeljka Korade Mirnics, Department of Pediatrics, nantly within B cells (Blk, Hck, Fgr, and Lck) (16–18) does not University of Pittsburgh, School of Medicine, Children’s Hospital of Pittsburgh, Pitts- burgh, PA 15213. Address questions regarding the microarray to Dr. Karoly Mirnics, produce a prominent phenotype (reviewed in Ref. 19). Although Departments of Psychiatry and Neurobiology, PittArray Microarray Core, University Lyn-deficient mice show normal B cell development within the of Pittsburgh, Pittsburgh, PA 15261. E-mail addresses: [email protected], bone marrow, the number of peripheral B cells in spleen, mesen- [email protected], [email protected] teric lymph nodes, and Peyer’s patches is reduced (15). Lyn defi- 3 Abbreviations used in this paper: BCR, B cell receptor; PTK, protein tyrosine ki- nase; GANP, germinal center-associated nuclear protein (germinal center-associated ciency is also associated with an increased number of immature B DNA primase); BDE, balanced differential expression; EF1␣, elongation factor 1␣. cells in the periphery (12, 15).

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 4134 Lyn-DEPENDENT EXPRESSION OF GANP

Recent advances in functional genomics highlighted the advan- activity. Reused membranes did not show sensitivity differences between tages of investigating complex gene expression patterns for the subsequent hybridizations (data not shown). discovery of novel signaling and disease-related pathways (20– Image acquiring and analysis. TIF files were analyzed using GeneSight 23). To determine putative downstream effectors associated with 4.0 software (BioDiscovery, Marina Del Rey, CA). After defining the Ϫ/Ϫ metagrid and subgrids, the spot pattern was inspected and manually ad- Lyn signaling in B cells, we compared Lyn and wild-type justed for pin performance and membrane stretch. For each probe spot, avian DT40 cells using custom-made chicken cDNA membrane mean/median signal intensity and mean/median local background measure- arrays (23). The analysis revealed a set of genes involved in B cell ments were performed. Spots were considered for further analysis if they development and signaling. One of these is germinal center-asso- produced a signal from Ͼ40% of the spot area. Array measurements were imported into Excel 2000 (Microsoft, Redmond, WA) and GeneSpring 4.1 ciated nuclear protein (germinal center-associated DNA primase) (Silicon Genetics, Redwood City, CA) for further analysis. (GANP), a B cell differentiation Ag. Expression of GANP, a 210- Data analysis. Data were normalized across the cDNA arrays. The 50th kDa phosphoprotein, is up-regulated in germinal centers of Ag- percentile of all measurements was used as a positive control for each immunized murine spleens (24). Through its C terminus, GANP sample; each measurement for each gene was divided by this synthetic binds to MCM3, a component of the DNA replication licensing positive control, assuming that this ratio was at least 10. This synthetic system that regulates initiation of DNA replication (25). CD40 control was the median of the gene’s expression values over all of the 502 samples. The bottom 10th percentile was used as a test for correct back- stimulation induces a phosphorylation (Ser )-dependent primase ground subtraction. The duplicate spots had to be within 40% of each other activity in the central region of GANP (26). Thus, GANP likely in intensity to be considered for further analysis. First, for each gene, the regulates DNA replication in activated centrocytes. A yeast two- intensities of duplicate spots were averaged. Then, the averages were cross- hybrid screen identified G5PR as a binding partner for GANP, and compared in a 3 ϫ 3 matrix, resulting in nine comparative values of bal- anced differential expression (BDE) for each of the 1440 genes. Based on Downloaded from because G5PR can recruit phosphatases PP5 and PP2A, the the nine BDEs, we generated a p value and performed a standard Z-score GANP/MCM3 complex may be regulated by phosphorylation dur- transformation (BDE gene Ϫ average BDE/SD). Finally, the nine BDE ing cell cycle progression (27). GANP expression may be partly values were averaged, determining the combined differential expression regulated by the transcription factor PU.1, because the GANP pro- value for each gene. Genes were considered to show differential expression moter has a PU.1 consensus sequence (28). Interestingly, Lyn and between conditions if they 1) showed an increased or decreased BDE value across seven of nine comparisons, 2) fell outside of 2 SDs from the mean PU.1 are found to be critical for both B cell and myeloid devel- combined differential expression value, and 3) reported a Z-score that ex- opment and signaling. In this study, we show that GANP and PU.1 ceeded 2 SDs from the mean in either direction. Hierarchical clustering was http://www.jimmunol.org/ expression are deficient in Lyn-deficient avian DT40 cells and performed using Cluster/Treeview software available from Stanford Uni- Lyn-deficient murine splenocytes. GANP promoter activity was versity (Palo Alto, CA). BDE values were log transformed, and the genes (but not the experiments) were clustered as described by Eisen et al. (31) dependent on both Lyn and the PU.1-sensitive site. Together, these using the complete clustering method. data suggest the presence of a Lyn3PU.13GANP pathway in B cells. Northern hybridization Total RNA was isolated from the cell lines and mouse spleen using TRIzol Materials and Methods (Life Technologies). The hybridization was performed using the Northern- Cell lines and Lyn-deficient mice Max system (Ambion, Austin, TX). Briefly, 20 ␮g of total RNA was by guest on September 28, 2021 loaded on a formamide gel, electrophoresed at 5 V/cm, transferred to a DT40 cell lines were generated by Dr. T. Kurosaki’s laboratory as previ- Bright Star nylon membrane (Ambion), and cross-linked by UV light ex- ously described (11). LynϪ/Ϫ mice were obtained from Drs. C. Lowell posure. Chicken probes were generated by enzymatic digestion of the orig- (University of California, San Francisco, CA) (15) and D. Linnekin (Na- inal clones made in Dr. J. Burnside’s laboratory. Mouse probes were gen- tional Cancer Institute, Frederick, MD) (29). erated using gene-specific primers in a standard PCR using normal spleen cDNA as a template. The product was labeled using a Random Primed DNA labeling kit (Roche, Basel, Switzerland) according to the manufac- Cell cultures turer’s instructions. To demonstrate comparable RNA loading, a 404-bp murine GAPDH probe was used. The 620-bp probe for chicken PU.1 was Wild-type and Lyn-deficient DT40 cells were grown in RPMI 1640 me- obtained by PCR of chicken cDNA from DT40 cells, with the forward dium with 10% FBS, 1% chicken serum, penicillin/streptomycin, and 50 primer, 5Ј-GAAGGATTTCCCCTCATTCC-3Ј, and the reverse primer, 5Ј- ␮M 2-ME as described previously (11). Cells were harvested 24 h after Ј ϫ 5 8 TGCGATTGCCTTTCTGTATG-3 . Northern blot was performed on plating at a density of 2 10 /ml. Approximately 10 cells were used for total RNA. isolation of total RNA and mRNA.

Isolation of mouse spleen B cells, immunization of mice, and cDNA array experiments immunohistochemistry Nitrocellulose cDNA arrays. The cDNAs on the arrays were selected from a chicken activated T cell library (30). Sequence data and information Mouse spleen B cells were enriched and cultured in RPMI 1640 medium regarding the identity of the clones can be found at the University of Del- as described previously (24, 32). Mice were immunized by multiple i.p. aware Chick EST Project website (http://chickest.udel.edu). Inserts were injections with SRBC (Colorado Serum, Denver, CO) as described previ- amplified by PCR using vector primers, and the quality and quantity of ously (24). Immunohistochemistry was done using Abs directed against 502 PCR products were evaluated by agarose gel electrophoresis (30). GANP or phospho-Ser -GANP as described previously (33). Isolation of RNA, labeling, and hybridization procedures. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to manu- Western blot facturer’s instructions, and mRNA extraction was performed using the Poly(A) Track mRNA isolation kit (Promega, Madison, WI). A total of 1 Wild-type and lynϪ/Ϫ mice were immunized with SRBC (Cappel, West ␮g of mRNA was used to perform first-strand cDNA synthesis using Chester, PA) by i.p. injections. Mice were sacrificed 7 days postinjection, [␣-32P]dCTP in an oligo(dT)-primed reverse transcription reaction (Life and spleens were harvested and snap frozen in methyl butane. Spleens were Technologies, Rockville, MD). A total of 20,000,000 cpm of radioactively homogenized in 500 ␮l of lysis buffer (1% Nonidet P-40, 25 mM Tris (pH labeled first-strand cDNA in 5-ml volume was hybridized overnight to each 7.5), 140 mM NaCl, 10 mM NaF, 5 mM EDTA, protease inhibitor mixture, of the membranes. The membranes were washed, exposed to phosphor and 1 mM Na vanadate) with a PowerGen 125 homogenizer to prepare screens, and scanned (Molecular Dynamics, Sunnyvale, CA). Hybridiza- whole-cell lysates. After centrifugation for 30 min at 13,000 rpm at 4°C, tions were repeated three times, using wild-type or Lyn-deficient DT40 supernatants were obtained and a modified Bradford protein assay (Bio- cultures, respectively, as starting material. After each of the hybridizations, Rad, Hercules, CA) was performed. A total of 150 ␮g of protein lysate per the membrane arrays were stripped, sealed, and exposed to x-ray film for sample was loaded and separated by SDS-PAGE on a 12.5% gel, trans- 48 h. None of the stripped membrane arrays demonstrated residual radio- ferred onto polyvinylidene difluoride membrane, and probed for PU.1 (1: The Journal of Immunology 4135

250), Lyn (1:1000), or Actin (1:1000) (all Abs from Santa Cruz Biotech- Results nology, Santa Cruz, CA). LynϪ/Ϫ DT40 cells demonstrate consistent gene expression differences GANP promoter reporter assay To determine the signaling pathways in which Lyn is involved, we The pGV-P constructs containing wild-type and PU.1/Ϫ134 mutated GANP promoter sites have been described elsewhere (28). DT40 and Lyn- have studied a chicken DT40 B cell line with a deletion of the Lyn deficient DT40 cells were cotransfected in triplicate with 10 ␮g of GANP gene (11). In contrast to the Src kinases expressed in normal B promoter constructs and 500 ng of pRLTK (Promega) containing the Re- cells, wild-type DT40 (DT40WT) cells express only Lyn PTK. As nilla luciferase gene as an internal control. The Dual-Luciferase Reporter a result, in the DT40 system, other Src PTKs family members Assay system (Promega) was used following the manufacturer’s protocol. Cells were harvested ϳ42 h posttransfection, pelleted, and washed once could not compensate for the absence of Lyn. Ϫ Ϫ with PBS, before being lysed with passive lysis buffer. The supernatant was Comparisons between the DT40WT and DT40Lyn / cells re- used to measure the luciferase activity using Analytical Luminescence vealed gene expression differences over the full range of signal ␮ Laboratory Monolight 2010 Luminometer. A total of 20 l of cell lysate intensity (data not shown); many of the transcript changes were was added to 100 ␮l of reagent LARII, and luciferase activity was mea- sured. To measure the Renilla luciferase activity, 100 ␮l of stop and glow identifiable even by visual inspection. The duplicate probe spots of buffer (Promega) was added to same tube. As a positive control, cells were the cDNA arrays showed consistent performance within all exper- transfected with vector pGL2 control (Promega) containing the luciferase iments for Ͼ99% of the genes investigated. The two cultured cell gene under SV40 promoter element. To compare the activity of the same lines did not show apparent growth rate or morphological differ- promoters in wild-type and Lyn-deficient cell lines, the Renilla luciferase activity (internal control) was used to normalize the luciferase activity ences, and, as expected, the majority of genes reported comparable values. expression between the two cell lines. Downloaded from

Table I. Significantly changed genes between the Lynϩ/ϩ and LynϪ/Ϫ DT40 cell lines: those decreased in the Lyn-deficient DT40 cell line

No. Accession No. Gene Function Z http://www.jimmunol.org/

1 AI979939 CD74* Signal transduction 6.10 2 AI980601 Uroguanylin Ionic homeostasis 6.09 3 AI980152 ␥-actin* Cytoskeletal organization 4.55 4 AI980603 Leukotriene hydrolase Arachidonic acid metabolism 4.05 5 AI979951 Farnesyl pyrophosphate synthetase Protein prenylation 3.33 6 BG712083 MHC IIi* Signal transduction 3.00 7 AI981844 GANP* Proliferation 2.72 8 AI980964 Transglutaminase* Cytoskeletal organization and proliferation 2.47 9 AI982328 EF1␣* Eukaryotic translation and elongation 2.40 10 AI979824 CD74* Signal transduction 2.35 by guest on September 28, 2021 11 BG625345 Prothymosin* Proliferation 2.34 12 AI981210 ICS-binding protein Transcription 2.17 13 AI980630 CD 79b Signal transduction 2.04 14 AI982328 EF1␣* Eukaryotic translation and elongation 2.03 15 AW239700 OBF-1* Transcription 2.00 16 AF151823 CGI-65 protein Unknown 1.87 17 AI981997 Ig HC* Signal transduction 1.85 18 AA495714 Rap 1A Signal transduction 1.85 19 AI981559 Calcineurin Phosphatase 1.84 20 n/a HuP1725 Unknown 1.71 21 AI981342 CD22* Signal transduction 1.62 22 AI979950 Stathmin* Cytoskeletal organization and proliferation 1.62 23 AI981250 NF-␬B-p100 subunit* Transcription factor 1.58 24 BG713270 MHC class 1 F10 ␣-chain precursor Cellular Ag 1.57 25 AI981484 Cathepsin C Endopeptidase 1.56 26 AI981994 KIAA 0323 Unknown 1.56 27 AI982155 Unknown Unknown 1.54 28 AA495704 Cathepsin B/Z Endopeptidase 1.49 29 AL117430 Hypothetical protein Unknown 1.49 30 AI981169 Y ␦ phosphatase receptor Unknown 1.42 31 AI980860 Tumor-associated MAGE-like protein Proliferation 1.34 32 AI979758 Elongation factor 2 Eukaryotic translation and elongation 1.31 33 AI982123 TCF1 Transcription factor 1.31 34 AI981057 Caveola-associated protein Unknown 1.28 35 AI981717 CEBP Transcription factor 1.27 36 BG713110 Nucleoside diphosphate kinase Proliferation and signal transduction 1.24 37 AI981613 Unknown protein Unknown 1.24 38 AI979763 Supt5h Transcription factor 1.23 39 AI979930 Chaperonin CCT ␦ Chaperonin 1.23 40 AI982328 EF1␣* Eukaryotic translation and elongation 1.20 41 AI980216 Act 1 Ser-Tre protein kinase 1.20 42 AI982000 KIAA 0905 ER-Golgi transport 1.18 43 AW239706 Ccr5 Chemokine receptor 1.16 44 AI982235 Bleomycin hydrolase Protease 1.16 45 AI981029 Unknown Unknown 1.16

.Verified by Northern hybridization ,ء 4136 Lyn-DEPENDENT EXPRESSION OF GANP

We identified 45 genes, the expression of which was decreased creased proliferation of peripheral B cells and poorly formed ger- in LynϪ/Ϫ DT40 cells relative to DT40WT cells, whereas only 2 minal centers within mesenteric lymph nodes and spleen (12, 15). genes with increased expression fulfilled the same criteria (Tables These findings, combined with our data, suggested that GANP I and II). Interestingly, many of the 45 genes with decreased ex- expression within splenic B cells might be at least partially re- pression were related to B cell response, regulation of transcrip- sponsible for the phenotype of the Lyn-deficient mice. Because the tion, proliferation, or cytoskeletal organization. expression level of GANP is low in normal B cells, we performed To uncover gene expression patterns across the comparisons, we SRBC stimulation of control and Lyn-deficient mice to induce ex- used hierarchical clustering. Clustering the log values of BDEs pression of GANP (24). As expected, staining of spleen sections across the nine comparisons revealed that most of our consistently with biotinylated reagent of peanut agglutinin (24) in wild-type changed genes showed up in a confined region of a cluster map animals showed formation of giant germinal centers with strongly (Fig. 1). Importantly, several different probes associated with the labeled GANPϩ cells (Fig. 4). However, spleen sections obtained genes elongation factor 1␣ (EF1␣) and CD74 were clustered as from LynϪ/Ϫ mice showed absence of germinal centers and GANP nearest neighbors, providing us with a further validation of our staining (Fig. 4A). An Ab raised against the serine phosphorylated experimental strategy. In addition to genes from Tables I and II, form of GANP showed the same staining pattern (Fig. 4B). In many of the genes found in the two clusters did not fulfill the wild-type mice, CD40 stimulation is known to result in GANP criteria we set forth for determining the most changed genes. These up-regulation in germinal centers (24). To test whether this mech- genes belonged to the classes of B cell response (MHC class I, anism is preserved in Lyn-deficient splenic B cells, we stimulated ␣2-HS glycoprotein) and proliferation (TGF, crn, 3-hydroxyacyl- B cells with anti-CD40 Ab. CD40 activation leads to rapid acti-

CoA dehydrogenase, zinc finger protein). Some of these expres- vation of Lyn (24, 34, 35). This stimulation led to a characteristic Downloaded from sion differences likely represent biologically significant changes proliferative response in wild-type cells, whereas proliferation was that are difficult to distinguish from experimental noise. Fig. 2 almost completely absent in LynϪ/Ϫ mouse B cells (Fig. 5). The depicts expression microarray demonstrated differences for 10 se- inability to up-regulate GANP and produce a proliferative B cell lected genes across the nine DT40 cell comparisons. These genes response in Lyn-deficient B cells suggests the existence of a Lyn were selected to represent the whole range of changed genes PTK-dependent, GANPϩ cell population, which may be essential

shown in Table I (CD74, CD22, GANP, NF-␬B, EF1␣, transglu- for germinal center formation and/or proliferation. http://www.jimmunol.org/ taminase, OBF-1, Ig HC, stathmin, prothymosin, and ␥-actin). In addition, we included two genes (MRJ of DNAJ, L5 ribosomal PU.1 expression and activity is Lyn PTK dependent subunit) that showed consistently decreased expression that did not Because GANP expression is dependent on PU.1 activity, the ef- meet our statistical criteria for inclusion in Tables I and II. Using fect of Lyn on PU.1 was studied. Northern blot analysis shows that Ϫ Ϫ a new isolate of wild-type and Lyn / DT40 cells, we verified the PU.1 expression was greater in wild-type than Lyn-deficient DT40 changes in gene expression for all of these genes by Northern cells or Lyn-deficient murine splenocytes (Fig. 6, A and B). Dif- hybridization (data not shown). ferences in PU.1 transcription factor activity between wild-type

Ϫ/Ϫ Ϫ/Ϫ and Lyn-deficient cells were also seen in luciferase promoter as- Lyn mouse spleen and Lyn DT40 cells exhibit reduced say. The promoter for GANP (Ϫ134) containing the luciferase by guest on September 28, 2021 GANP expression reporter construct into wild-type and Lyn-deficient DT40 cells. A We hypothesized that some Lyn functions are conserved across 2-fold greater activity in wild-type cells was observed. Confirming species and experimental systems, and that a portion of the ob- that GANP promoter activity was at least partly due to its Lyn- served gene expression changes in the LynϪ/Ϫ DT40 cells will be sensitive PU.1 binding site, we observed that wild-type enhanced observed in LynϪ/Ϫ mice. To test this hypothesis, we have chosen activity was reduced by 67% when the PU.1 binding site was mu- to examine the expression of five genes (GANP, PHB2, transglu- tated (Fig. 6C). Interestingly, PU.1-sensitive luciferase activity taminase, pyruvate kinase, and EF1␣) in the spleen total RNA of was diminished by the same degree in Lyn-deficient DT40 cells. the LynϪ/Ϫ mice. Besides their differential expression in LynϪ/Ϫ Together, these studies suggest that Lyn affects GANP expression DT40 and DT40WT cells, these genes were chosen based upon by enhancing both expression and activity of PU.1. their importance for B cell signaling or for their unknown role in the Lyn signaling cascade. Using total RNA isolated from Lyn- Discussion deficient mouse spleen and Northern hybridization, we were able Lyn is involved in multiple signaling processes. We used cDNA to verify expression changes for GANP, but not for EF1␣, pyru- array analysis to identify previously unknown Lyn-dependent vate kinase, and transglutaminase (data not shown). A Northern functional signaling cascades that are conserved across species and blot performed showed that GANP expression was markedly re- experimental systems. This experimental design helped us to as- duced in murine splenocytes from Lyn-deficient mice (Fig. 3). sess the overall involvement of Lyn in B cell signaling, as well as

Ϫ/Ϫ to identify a subset of signaling events that uniquely depend on GANP-dependent proliferation is absent in the Lyn mice preserved Lyn function. Although GANP has not been previously implicated in Lyn sig- The changes identified in the LynϪ/Ϫ DT40 cells argue that Lyn naling, it is involved in proliferation of B cells and formation of PTK is involved in a multitude of cellular processes including germinal centers (24). Interestingly, Lyn-deficient mice show de- BCR-signaling (CD74, CD22, Ig ␣ H chain, Ig-associated ␤, and

Table II. Significantly changed genes between the Lynϩ/ϩ and LynϪ/Ϫ DT40 cell lines: those increased in the Lyn-deficient DT40 line

No. Accession No. Gene Function Z

1 AB011167 Unknown Unknown Ϫ1.19 2 NP_003302 Tumor-suppressing subtransferable 3 Unknown Ϫ1.19 The Journal of Immunology 4137 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Hierarchical clustering of LynϪ/Ϫ and Lynϩ/ϩ gene expression differences. The right panel dendrogram is generated using the cDNA array comparison data in Cluster/Treeview by the method of Eisen et al. (31). Note that several regions show clustering of consistent increases and decreases between the two compared conditions (Lynϩ/ϩ vs LynϪ/Ϫ). Two such regions, depicted by color bars, are enlarged: the left panel represents clusters of genes that reported consistently decreased expression in the Lyn-deficient cell lines, whereas the middle panel corresponds to clusters encompassing genes that reported the most consistent increases. Note that, within the left panel, the software clustered three different probes of EF1␣ and three MHC class II genes as nearest neighbors, further demonstrating the power of this analytical approach. 4138 Lyn-DEPENDENT EXPRESSION OF GANP

FIGURE 2. Differential expression of 13 selected genes. Logged BDE values are plotted on the y-axis across the nine comparisons (C1–C9) on x-axis. Note that most of the comparisons report consistent decrease for these 13 genes.

CD79b), proliferation (GANP, tumor-associated MAGE like, nu- cleoside diphosphate kinase, stathmin, prothymosin, and transglu- Downloaded from taminase), control of transcription (transcription factors, EF1␣, cleavage and polyadenylation specificity factor, CEBP, TCF1, OBF-1, ICS BP), immunity and inflammation response (NF-␬B), and cytoskeletal organization (␥-actin, transglutaminase, chapero- nin CCT ␦, stathmin). Interestingly, compared with the number of genes that are down- regulated in the absence of Lyn, there are only a few genes that http://www.jimmunol.org/ showed consistently increased expression as a result of Lyn defi- ciency. This is somewhat surprising because Src PTKs can activate or inhibit immunoreceptor-mediated signal transduction depending on the substrates they phosphorylate (36–38). However, we mea- sured steady-state changes in unstimulated DT40 cells, and gene expression increases as a result of Lyn deficiency may become obvious only when these cells are activated. However, despite the

enormous discovery potential of high-throughput transcriptome by guest on September 28, 2021 profiling, general limitations of the microarray datasets include both type I and type II errors. In our experimental series, for the most robustly changed genes, type I error was limited, and the critical data were verified in follow-up experiments. However, we expect that there are further (and potentially significant) expression changes that were not uncovered by our analysis (type II error). Furthermore, many important biological processes occur at the protein level, and these events were not the focus of our investigation.

Lyn deficiency leads to altered expression of genes forming the BCR complex We found significantly altered expression of at least five genes that are involved in the formation of BCR complex. Ig H chain is the

FIGURE 4. LynϪ/Ϫ mouse germinal centers stimulated by SRBC fail to induce GANP expression. A, GANP staining. Sections of LynϪ/Ϫ and Lynϩ/Ϫ mouse spleen germinal centers stained with peanut agglutinin (PNA) (top) and anti-GANP Ab (bottom) after SRBC stimulation. Note FIGURE 3. Northern blot of murine splenocytes for GANP expression. that heterozygous mice (left panels) proliferated and up-regulated GANP, Splenocytes were purified from lynϪ/Ϫ mice or their homozygous wild- whereas in Lyn-deficient mice (right panels), this response was absent (RP, type littermates. Total RNA was harvested, and Northern blotting was per- red pulp; WP, white pulp; GC, germinal center). B, Phospho-Ser502-GANP formed for expression of murine GANP, with GAPDH cDNA used as a staining. Sections were stained for Ab against the serine phosphorylated control for RNA loaded. form of GANP, as described elsewhere (24). The Journal of Immunology 4139

FIGURE 5. LynϪ/Ϫ and Lynϩ/Ϫ B cells respond differently to CD40 stimulation. Anti-GANP staining of unstimulated and CD40-stimulated B cells from heterozygous and LynϪ/Ϫ mice. Note that Lyn-deficient B cells Downloaded from do not up-regulate GANP in response to CD40 stimulation.

major subunit of Ag-binding component, CD79b is the intracellu- lar signaling component of the BCR, CD22 is a BCR coreceptor, http://www.jimmunol.org/ whereas OBF-1 is a transcription factor (39). Although it is well established that Lyn phosphorylates CD79b and CD22, it has not been reported that Lyn affects their expression (40). Furthermore, CD74 functions as a chaperone, transporting subunits of the BCR to the plasma membrane. These decrements in the expression of multiple related genes are highly suggestive of an impaired regu- lation/assembly of the BCR and may result in an altered BCR signaling. OBF-1 is a transcription factor, most abundantly ex-

pressed in B cells (41). Mice deficient in OBF-1 have reduced by guest on September 28, 2021 numbers of mature B cells, a severe reduction in the number of recirculating B cells, and poor germinal center formation (42). Al- together, these data suggest that Lyn controls BCR expression, assembly, and signaling in part through OBF-1 (4, 43). Lyn has been described as a double-edged kinase that can promote or in- hibit growth (14). B cell apoptosis can be induced through CD40/ FIGURE 6. Lyn regulates level and activity of the transcription factor CD40 ligand or through BCR cross-linking (44, 45). Lyn-mediated PU.1. A, Northern blot analysis of PU.1 in DT40 cells. Comparison of phosphorylation of CD22 also serves to control growth. These pro- wild-type and Lyn-deficient DT40 cells show that there are decreased lev- cesses may also occur simultaneously or sequentially, reducing B els of PU.1 in Lyn-deficient cells compared with wild-type cells. B, West- cell viability in a complex process. Thus, this microarray analysis ern blot analysis of PU.1 in murine splenocytes. Comparison of wild-type suggests that a PTK can affect a molecule through both posttrans- and Lyn-deficient murine splenocytes show that there are decreased protein lational and transcriptional means. levels of PU.1 (indicated by arrow) in Lyn-deficient cells. In lower panel, absence of Lyn is demonstrated in Lyn-deficient murine splenocytes. Com- parable levels of protein loading are shown by actin blotting in the middle Some Lyn PTK functions may not be unique panel. Protein lysates were prepared from splenocytes from individual Not all of the gene expression changes described above need be mice (indicated by number). As controls, protein lysates were prepared uniquely dependent on preserved Lyn function. Although DT40 from the murine cell lines Ba/F3 and 32D. C, GANP promoter activity in cells do not express Src kinases other then Lyn (11), both DT40 wild-type and Lyn-deficient DT40 cells. Wild-type and Lyn-deficient cell line and mammalian B cells contain BTK and Syk (46). We DT40 cells were transfected with constructs containing the GANP pro- Ϫ Ϫ moter and the luciferase reporter gene. Wild-type and Lyn-deficient cells hypothesize that gene expression differences found in Lyn / were also transfected with the construct containing a mutated PU.1 binding DT40 cells but not in the mouse Lyn-deficient spleen isolate (e.g., site (*PU.1), as described elsewhere. Values are expressed as relative lu- ␣ EF1 , transglutaminase) are due to the redundant effects of other ciferase units (mean Ϯ SD). Src kinase members. This redundancy is clearly present in B cells, although it may not be characteristic of other cell populations and tissues in the Lyn-deficient mice. The exact phenotypic deficits across tissues may be related to the tissue-specific coexpression Lyn PTK has a unique function in GANP-mediated proliferation pattern and functional redundancy of different Src PTK family Based on our findings and previous studies, Lyn also has a unique members. Some of the transcriptome changes may also be specific function in B cell physiology that is conserved across different to species. However, the altered GANP expression across both species. Changes in GANP expression in both chicken and mouse avian and mammalian lymphoid tissues strengthens the physiolog- LynϪ/Ϫ models suggest that germinal center proliferation is a ma- ical relevance of our findings. jor and conserved function of Lyn PTK. Indeed, GANP has been 4140 Lyn-DEPENDENT EXPRESSION OF GANP previously suggested as an essential molecule for the formation of 9. Corey, S. J., and S. M. Anderson. 1999. Src-related protein tyrosine kinases in germinal centers (24). IgM and CD40 stimulation both up-regulate hematopoiesis. Blood 93:1. Ϫ/Ϫ 10. Brown, W. R., S. J. Hubbard, C. Tickle, and S. A. Wilson. 2003. The chicken as expression of GANP, and Lyn B cells do not respond to IgM a model for large-scale analysis of vertebrate gene function. Nat. Rev. Genet. stimulation. Our data show that stimulation by CD40 also fails to 4:87. induce GANP response in LynϪ/Ϫ B cells, thus confirming previ- 11. Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, and T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor- ous reports (47, 48). This unresponsiveness is the most likely coupled Ca2ϩ mobilization through distinct pathways. EMBO J. 13:1341. underlying cause of the lack of proliferative response in the Lyn- 12. Hibbs, M. L., D. M. Tarlinton, J. Armes, D. Grail, G. Hodgson, R. Maglitto, S. A. Stacker, and A. R. Dunn. 1995. Multiple defects in the immune system of deficient mice and may contribute to some of their phenotypic Lyn-deficient mice, culminating in autoimmune disease. Cell 83:301. deficits (no Peyer’s patches, reduced number of mature B cells in 13. Nishizumi, H., I. Taniuchi, Y. Yamanashi, D. Kitamura, D. Ilic, S. Mori, spleen). Furthermore, SRBC stimulation does not lead to a prolif- T. Watanabe, and T. Yamamoto. 1995. Impaired proliferation of peripheral B ϩ cells and indication of autoimmune disease in lyn-deficient mice. Immunity erating population of GANP cells in splenic germinal centers. 3:549. This absence of proliferation supports a physiological role for Lyn- 14. Nishizumi, H., K. Horikawa, I. Mlinaric-Rascan, and T. Yamamoto. 1998. A dependent GANP expression as part of B cell response. double-edged kinase Lyn: a positive and negative regulator for antigen receptor- mediated signals. J. Exp. Med. 187:1343. 15. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, and C. A. Lowell. 1997. Lyn affects PU.1 Characterization of the B lymphocyte populations in Lyn-deficient mice and the We have previously shown that GANP expression is partially con- role of Lyn in signal initiation and down-regulation. Immunity 7:69. 16. Link, D. C., and M. Zutter. 1995. The proto-oncogene c-fgr is expressed in trolled by a critical PU.1 binding site in its promoter region (28). normal mantle zone B lymphocytes and is developmentally regulated during my- In this study, we show that the presence of Lyn regulates PU.1 elomonocytic differentiation in vivo. Blood 85:472. expression and PU.1-dependent activity of the GANP promoter. 17. Taguchi, T., N. Kiyokawa, N. Sato, M. Saito, and J. Fujimoto. 2000. Character- Downloaded from istic expression of Hck in human B-cell precursors. Exp. Hematol. 28:55. Transient transfection of the GANP promoter showed it to be more 18. Wechsler, R. J., and J. G. Monroe. 1995. src-family tyrosine kinase p55fgr is active in wild-type DT40 cells than Lyn-deficient DT40 cells. expressed in murine splenic B cells and is activated in response to antigen re- Moreover, mutation of the PU.1 consensus binding site in the ceptor cross-linking. J. Immunol. 154:3234. 19. Korade-Mirnics, Z., and S. J. Corey. 2000. Src kinase-mediated signaling in GANP promoter abrogated the differential response. Although no leukocytes. J. Leukocyte Biol. 68:603. reports have yet linked PU.1 with Lyn, both share similar tissue 20. Mirnics, K. 2001. Microarrays in brain research: the good, the bad and the ugly. Nat. Rev. Neurosci. 2:444. distribution (primarily B lymphocytes and myeloid-derived gran- http://www.jimmunol.org/ 21. Mirnics, K., F. A. Middleton, D. A. Lewis, and P. Levitt. 2001. Analysis of ulocytes, eosinophils, macrophages, and dendritic cells) (49, 50). complex brain disorders with gene expression microarrays: schizophrenia as a Further review of promoter sites of other genes identified in this disease of the synapse. Trends Neurosci. 24:479. Lyn microarray screen, such as CD79b and CD22 (51), revealed 22. Mirnics, K., D. A. Lewis, and P. Levitt. 2001. DNA microarrays in human brain disorders. In Methods in Genomic Neuroscience. H. Chin and S. Moldin, eds. putative PU.1 binding sites. PU.1 expression itself is autoregulated CRC, London, p. 171. (52), which would be consistent with our finding that PU.1 ex- 23. Neiman, P. E., A. Ruddell, C. Jasoni, G. Loring, S. J. Thomas, K. A. Brandvold, R. Lee, J. Burnside, and J. Delrow. 2001. Analysis of gene expression during myc pression was decreased in Lyn-deficient DT40 cells. Furthermore, oncogene-induced lymphomagenesis in the bursa of Fabricius. Proc. Natl. Acad. PU.1 activity is enhanced by its phosphorylation, which may be Sci. USA 98:6378. mediated by Akt or Jnk (53, 54). Both of these serine kinases are 24. Kuwahara, K., M. Yoshida, E. Kondo, A. Sakata, Y. Watanabe, E. Abe,

Y. Kouno, S. Tomiyasu, S. Fujimura, T. Tokuhisa, et al. 2000. A novel nuclear by guest on September 28, 2021 regulated by downstream events dependent on Lyn (55–57). To- phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and gether, these studies demonstrate the utility of microarray analysis associated with MCM3, a protein essential for DNA replication. Blood 95:2321. in identifying a small subset of physiologically relevant genes and 25. Chong, J. P., P. Thommes, and J. J. Blow. 1996. The role of MCM/P1 proteins in the licensing of DNA replication. Trends Biochem. Sci. 21:102. transcription factor across species, which were not previously con- 26. Kuwahara, K., S. Tomiyasu, S. Fujimura, K. Nomura, Y. Xing, N. Nishiyama, sidered to be part of a signaling pathway. Based on these studies, M. Ogawa, S. Imajoh-Ohmi, S. Izuta, and N. Sakaguchi. 2001. Germinal center- we propose a Lyn3PU.13GANP pathway in developing B cells. associated nuclear protein (GANP) has a phosphorylation-dependent DNA-pri- mase activity that is up-regulated in germinal center regions. Proc. Natl. Acad. Sci. USA 98:10279. Acknowledgments 27. Kono, Y., K. Maeda, K. Kuwahara, H. Yamamoto, E. Miyamoto, K. Yonezawa, We thank Joshua Lovelock and Deborah Holligshead for help with cDNA K. Takagi, and N. Sakaguchi. 2002. MCM3-binding GANP DNA-primase is associated with a novel phosphatase component G5PR. Genes Cells 7:821. array data analysis. We are grateful to Drs. Diana Linnekin and Clifford 28. El-Gazzar, M. A., K. Maeda, H. Nomiyama, M. Nakao, K. Kuwahara, and Lowell for providing Lyn-deficient mice. We are also thankful to N. Sakaguchi. 2001. PU.1 is involved in the regulation of B lineage-associated Dr. Nina F. Schor and Dana Murphy for providing help with data verifi- and developmental stage-dependent expression of the germinal center- associated cation and experiments, and for valuable comments on the manuscript. DNA primase GANP. J. Biol. Chem. 276:48000. 29. O’Laughlin-Bunner, B., N. Radosevic, M. L. Taylor, Shivakrupa, C. DeBerry, D. D. Metcalfe, M. Zhou, C. Lowell, and D. Linnekin. 2001. Lyn is required for References normal stem cell factor-induced proliferation and chemotaxis of primary hema- 1. Cambier, J. C., and J. T. Ransom. 1987. Molecular mechanisms of transmem- topoietic cells. Blood 98:343. brane signaling in B lymphocytes. Annu. Rev. Immunol. 5:175. 30. Tirunagaru, V. G., L. Sofer, J. Cui, and J. Burnside. 2000. An expressed sequence 2. Kurosaki, T. 1998. Molecular dissection of B cell antigen receptor signaling tag database of T-cell-enriched activated chicken splenocytes: sequence analysis (review). Int. J. Mol. Med. 1:515. of 5251 clones. Genomics 66:144. 3. Kurosaki, T. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. 31. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein. 1998. Cluster anal- Rev. Immunol. 17:555. ysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 4. Burkhardt, A. L., M. Brunswick, J. B. Bolen, and J. J. Mond. 1991. Anti-immu- 95:14863. noglobulin stimulation of B lymphocytes activates src-related protein-tyrosine 32. Igarashi, H., N. Kuwata, K. Kiyota, K. Sumita, T. Suda, S. Ono, S. R. Bauer, and kinases. Proc. Natl. Acad. Sci. USA 88:7410. N. Sakaguchi. 2001. Localization of recombination activating gene 1/green flu- 5. Campbell, M. A., and B. M. Sefton. 1992. Association between B-lymphocyte orescent protein (RAG1/GFP) expression in secondary lymphoid organs after membrane immunoglobulin and multiple members of the Src family of protein immunization with T-dependent antigens in rag1/gfp knockin mice. Blood tyrosine kinases. Mol. Cell. Biol. 12:2315. 97:2680. 6. Kurosaki, T., M. Takata, Y. Yamanashi, T. Inazu, T. Taniguchi, T. Yamamoto, 33. Abe, E., K. Kuwahara, M. Yoshida, M. Suzuki, H. Terasaki, Y. Matsuo, and H. Yamamura. 1994. Syk activation by the Src-family tyrosine kinase in the E. I. Takahashi, and N. Sakaguchi. 2000. Structure, expression, and chromosomal B cell receptor signaling. J. Exp. Med. 179:1725. localization of the human gene encoding a germinal center-associated nuclear 7. DeFranco, A. L., J. D. Richards, J. H. Blum, T. L. Stevens, D. A. Law, protein (GANP) that associates with MCM3 involved in the initiation of DNA V. W. Chan, S. K. Datta, S. P. Foy, S. L. Hourihane, M. R. Gold, et al. 1995. replication. Gene 255:219. Signal transduction by the B-cell antigen receptor. Ann. NY Acad. Sci. 766:195. 34. Faris, M., F. Gaskin, J. T. Parsons, and S. M. Fu. 1994. CD40 signaling pathway: 8. Corey, S. J., P. M. Dombrosky-Ferlan, S. Zuo, E. Krohn, A. D. Donnenberg, anti-CD40 monoclonal antibody induces rapid dephosphorylation and phosphor- P. Zorich, G. Romero, M. Takata, and T. Kurosaki. 1998. Requirement of Src ylation of tyrosine-phosphorylated proteins including protein tyrosine kinase kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating Lyn, Fyn, and Syk and the appearance of a 28-kD tyrosine phosphorylated pro- factor. J. Biol. Chem. 273:3230. tein. J. Exp. Med. 179:1923. The Journal of Immunology 4141

35. Ren, C. L., T. Morio, S. M. Fu, and R. S. Geha. 1994. Signal transduction via 46. Satterthwaite, A. B., Z. Li, and O. N. Witte. 1998. Btk function in B cell devel- CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and opment and response. Semin. Immunol. 10:309. phosphorylation of phospholipase C␥2. J. Exp. Med. 179:673. 47. Kato, J., N. Motoyama, I. Taniuchi, H. Takeshita, M. Toyoda, K. Masuda, and 36. Malbec, O., D. C. Fong, M. Turner, V. L. Tybulewicz, J. C. Cambier, T. Watanabe. 1998. Affinity maturation in Lyn kinase-deficient mice with defec- W. H. Fridman, and M. Daeron. 1998. Fc⑀ receptor I-associated lyn-dependent tive germinal center formation. J. Immunol. 160:4788. phosphorylation of Fc␥ receptor IIB during negative regulation of mast cell ac- 48. Wang, J., T. Koizumi, and T. Watanabe. 1996. Altered antigen receptor signaling tivation. J. Immunol. 160:1647. and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice. J. Exp. 37. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science Med. 184:831. 290:84. 49. Nerlov, C., and T. Graf. 1998. PU.1 induces myeloid lineage commitment in 38. Smith, K. G., D. M. Tarlinton, G. M. Doody, M. L. Hibbs, and D. T. Fearon. multipotent hematopoietic progenitors. Genes Dev. 12:2403. 1998. Inhibition of the B cell by CD22: a requirement for Lyn. J. Exp. Med. 50. Scott, E. W., M. C. Simon, J. Anastasi, and H. Singh. 1994. Requirement of 187:807. transcription factor PU.1 in the development of multiple hematopoietic lineages. 39. Krapp, A., and M. Strubin. 1999. B-Cell coactivator OBF-1 exhibits unusual Science 265:1573. transcriptional properties and functions in a DNA-bound Oct-1-dependent fash- 51. Andersson, K. B., K. E. Draves, D. M. Magaletti, S. Fujioka, K. L. Holmes, ion. Mol. Cell. Biol. 19:4247. C. L. Law, and E. A. Clark. 1996. Characterization of the expression and gene promoter of CD22 in murine B cells. Eur. J. Immunol. 26:3170. 40. Cornall, R. J., J. G. Cyster, M. L. Hibbs, A. R. Dunn, K. L. Otipoby, E. A. Clark, 52. Chen, H., D. Ray-Gallet, P. Zhang, C. J. Hetherington, D. A. Gonzalez, and C. C. Goodnow. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 D. E. Zhang, F. Moreau-Gachelin, and D. G. Tenen. 1995. PU.1 (Spi-1) auto- are limiting elements of a biochemical pathway regulating BCR signaling and regulates its expression in myeloid cells. Oncogene 11:1549. selection. Immunity 8:497. 53. Rieske, P., and J. M. Pongubala. 2001. AKT induces transcriptional activity of 41. Strubin, M., J. W. Newell, and P. Matthias. 1995. OBF-1, a novel B cell-specific PU.1 through phosphorylation-mediated modifications within its transactivation coactivator that stimulates immunoglobulin promoter activity through association domain. J. Biol. Chem. 276:8460. with octamer-binding proteins. Cell 80:497. 54. Behre, G., A. J. Whitmarsh, M. P. Coghlan, T. Hoang, C. L. Carpenter, 42. Schubart, D. B., A. Rolink, M. H. Kosco-Vilbois, F. Botteri, and P. Matthias. D. E. Zhang, R. J. Davis, and D. G. Tenen. 1999. c-Jun is a JNK-independent 1996. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune re- coactivator of the PU.1 transcription factor. J. Biol. Chem. 274:4939. sponse and germinal centre formation. Nature 383:538. 55. Dong, F., and A. C. Larner. 2000. Activation of Akt kinase by granulocyte col- Downloaded from 43. Yamanashi, Y., T. Kakiuchi, J. Mizuguchi, T. Yamamoto, and K. Toyoshima. ony-stimulating factor (G-CSF): evidence for the role of a tyrosine kinase activity 1991. Association of B cell antigen receptor with protein tyrosine kinase Lyn. distinct from the Janus kinases. Blood 95:1656. Science 251:192. 56. Li, H. L., W. W. Davis, E. L. Whiteman, M. J. Birnbaum, and E. Pure. 1999. The 44. Donjerkovic, D., and D. W. Scott. 2000. Activation-induced cell death in B lym- tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein phocytes. Cell Res. 10:179. kinase Akt/PKB in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:6890. 45. Hagiyama, H., T. Adachi, T. Yoshida, T. Nomura, N. Miyasaka, T. Honjo, and 57. Jiang, A., A. Craxton, T. Kurosaki, and E. A. Clark. 1998. Different protein T. Tsubata. 1999. Signaling through the antigen receptor of B lymphocytes ac- tyrosine kinases are required for B cell antigen receptor-mediated activation of

tivates a p53-independent pathway of c-Myc-induced apoptosis. Oncogene extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mi- http://www.jimmunol.org/ 18:4091. togen-activated protein kinase. J. Exp. Med. 188:1297. by guest on September 28, 2021