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CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)- γ 2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase This information is current as D-Dependent Signaling Cascade of September 23, 2021. Miguel E. Moreno-García, Lucia N. López-Bojórques, Alejandro Zentella, Lisa A. Humphries, David J. Rawlings and Leopoldo Santos-Argumedo J Immunol 2005; 174:2687-2695; ; Downloaded from doi: 10.4049/jimmunol.174.5.2687 http://www.jimmunol.org/content/174/5/2687 http://www.jimmunol.org/ References This article cites 66 articles, 39 of which you can access for free at: http://www.jimmunol.org/content/174/5/2687.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 23, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-␥2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade1 Miguel E. Moreno-Garcı´a,*† Lucia N. Lo´pez-Bojo´rques,‡ Alejandro Zentella,‡ Lisa A. Humphries,§ David J. Rawlings,§ and Leopoldo Santos-Argumedo2† The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-␥2 (PLC-␥2), functions to gen- erate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no Downloaded from detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-␥2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-␥2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) ␤-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol- dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-␬B activation, http://www.jimmunol.org/ and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)- metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, I␬B-␣ degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-␥2-independent, B lymphocyte activation. The Journal of Im- munology, 2005, 174: 2687–2695. by guest on September 23, 2021 he CD38 is a 42-kDa type II transmembrane glycoprotein CD38, however, also displays activity as a cell surface receptor. expressed in many tissues, including most hemopoietic Activation through CD38 with agonistic Abs triggers a variety of T cell lineages (1–3). CD38 was initially identified as an responses including: cell proliferation, rescue from apoptosis, in- ectoenzyme able to hydrolyze ␤-nicotinamide adenine dinucle- ducible protein tyrosine phosphorylation, a low level increase in otide to cyclic adenosine-diphosphoribose, adenosine diphospho- intracellular calcium, and the expression of several surface mole- ribose, and nicotinamide (4, 5). It is known that cyclic adenosine- cules including MHC-II and CD69 (7–9). diphosphoribose triggers calcium mobilization from the Although preliminary investigations of the CD38 receptor endoplasmic reticulum (ER)3 through ryanodine receptors (6). have revealed varied functions, the signaling events mediated by CD38 engagement remain unclear. Indeed, while the prolif- erative response of mature splenic B cells to CD38 has been *Departments of Cell Biology and †Molecular Biomedicine, Centro de Investigacio´n shown to require both Src (Lyn and Fyn) (10) and Tec family y Estudios Avanzados, Mexico D.F. Mexico; ‡Department of Cell Biology, Instituto kinases (Btk) (11, 12), their downstream substrates remain un- de Fisiologı´a Celular, Universidad Nacional Autónoma de México, Mexico D.F., Mexico; and §Department of Pediatrics, University of Washington School of Medi- known. A well-characterized target of Btk in BCR-mediated cine, Seattle, WA 98195 signaling is the phosphoinositide-phospholipase C-␥2 (PI-PLC- Received for publication December 19, 2003. Accepted for publication December ␥2). Btk is essential for the activation of PLC-␥2 (13–15), and 13, 2004. these enzymes work in concert to produce downstream signals The costs of publication of this article were defrayed in part by the payment of page required for B cell survival, differentiation, and proliferation. charges. This article must therefore be hereby marked advertisement in accordance Once activated, PLC-␥2 mediates hydrolysis of phosphatidyl- with 18 U.S.C. Section 1734 solely to indicate this fact. inositol 4,5-bisphosphate (PIP ), leading to the sustained pro- 1 This work was supported by grants to L.S.-A. from Consejo Nacional de Ciencia y 2 Tecnologı´a (Conacyt), Me´xico (28093N and 33497N). D.J.R. is the recipient of a duction of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol McDonnell Scholar Award, a Leukemia and Lymphoma Society Scholar Award, and (DAG) required for an initial intracellular calcium release and the Joan J. Drake Grant for Excellence in Cancer Research. This work was partially protein kinase C (PKC) activation, respectively (16). supported by National Institutes of Health Grants HD37091, CA81140, AI38348, and AI33617, and the American Cancer Society. Previous studies, however, have suggested that CD38-mediated 2 Address correspondence and reprint requests to Dr. Leopoldo Santos-Argumedo, intracellular calcium flux is dependent on extracellular calcium Departamento de Biomedicina Molecular, Centro de Investigacio´n y Estudios Avan- sources and occurs by a mechanism that is independent of inositol zados del I.P.N., Apartado Postal 14-740, cp 07360, Me´xico D.F. Me´xico. E-mail address: [email protected] 3 Abbreviations used in this paper: ER, endoplasmic reticulum; DAG, diacylglycerol; phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; IP3, inositol-1,4,5 trisphosphate; PC, phosphatidylcholine; PI-PLC, phosphoinositide- PLC, phospholipase C; PLD, phospholipase D; PY, phosphotyrosine; WT, wild type. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 2688 CD38 SIGNALING USES PC-PLC, PLD, AND PKC IN B LYMPHOCYTES phosphate generation and internal calcium-store release (8, 9). Al- IgG for 0.5, 1, 5, and 10 min. Cells were lysed in buffer containing 150 mM though these observations suggest that PLC-␥2 function may not NaCl, 10 mM Tris-base, pH 7.2, 2 mM Na3VO4, 0.4 mM EDTA, 10 mM ␮ be required for CD38 signaling, they are seemingly in conflict with NaF, 1 mM PMSF, 5 g/ml aprotinin and leupeptin, and 1% Nonidet P-40. PLC-␥2 was immunoprecipitated with an anti-PLC-␥2 Ab (Santa Cruz the requirement of Btk in CD38 signaling. Biotechnology) for1hat4°C, and Ag-Ab complexes were precipitated To begin to address this apparent paradox, we evaluated the using protein G-Sepharose (Zymed Laboratories). Proteins were resolved participation of PLC-␥2 in CD38 signaling in murine splenic B by SDS-PAGE and transferred to nitrocellulose. For phosphotyrosine (PY) cells. Our studies show that CD38-dependent proliferation is un- detection, the membrane was probed with the anti-phosphotyrosine Ab 4G10-HRP (Upstate Biotechnology) and developed by ECL-Plus (Amer- affected by the addition of a PLC-specific inhibitor (U73122) and sham Biosciences). The membrane was stripped and reanalyzed with the that cross-linking of the CD38 receptor fails to induce IP3 gener- anti-PLC-␥2 Ab, followed by an anti-rabbit-HRP Ab (DakoCytomation). ation. Consistent with these results, there was a loss of receptor- For the detection of the phosphorylated tyrosine residue 759 of PLC-␥2 dependent, functional PLC-␥2 phosphorylation, as evidenced by (pY759), B cells were stimulated for 1, 5, and 10 min with anti-CD38, the absence of phosphorylation at PLC-␥2 Y759, a key regulatory anti-IgM, and rat IgG, and pY759 was detected by Western
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