Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation This information is current as Yuko Kawakami, Jiro Kitaura, Anne B. Satterthwaite, of September 25, 2021. Roberta M. Kato, Koichi Asai, Stephen E. Hartman, Mari Maeda-Yamamoto, Clifford A. Lowell, David J. Rawlings, Owen N. Witte and Toshiaki Kawakami J Immunol 2000; 165:1210-1219; ; doi: 10.4049/jimmunol.165.3.1210 Downloaded from http://www.jimmunol.org/content/165/3/1210 References This article cites 75 articles, 45 of which you can access for free at: http://www.jimmunol.org/content/165/3/1210.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation1 Yuko Kawakami,* Jiro Kitaura,* Anne B. Satterthwaite,† Roberta M. Kato,‡ Koichi Asai,* Stephen E. Hartman,* Mari Maeda-Yamamoto,§ Clifford A. Lowell,¶ David J. Rawlings,‡ Owen N. Witte,†ʈ and Toshiaki Kawakami2* Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (Fc⑀RI). In this study, we have made the following observations on growth properties and Fc⑀RI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, Fc⑀RI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-␥1 and C-␥2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, Fc⑀RI-induced elevation of intracellular Ca2؉ concentrations and Downloaded from activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-␣ and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase C␣ and protein kinase C␤II, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun http://www.jimmunol.org/ N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components. The Journal of Immunology, 2000, 165: 1210–1219. ross-linking of IgE-bound high-affinity IgE receptors the ␤ subunit in resting cells through interaction of the N-terminal (Fc⑀RI)3 on mast cells by Ag leads to the activation of unique region of Lyn with the C-terminal cytoplasmic domain of C mast cells culminating in the release of a panel of proin- the ␤ subunit (3, 4). Lyn is activated by transphosphorylation upon flammatory mediators. Mast cell activation triggers reactions of Fc⑀RI cross-linking (5). Activated Lyn phosphorylates the tyrosine by guest on September 25, 2021 immediate hypersensitivity (1). Fc⑀RI is composed of an IgE-bind- residues in the immunoreceptor tyrosine-based activation motifs ing ␣ subunit, a four-transmembrane, signal-amplifying ␤ subunit, (ITAM) of the cytoplasmic regions of the ␤ and ␥ subunits (6). and a disulfide-bonded pair of ␥ subunits (reviewed in Ref. 2). The Phosphorylated ITAMs of the ␤ and ␥ subunits recruit Lyn and Src family protein tyrosine kinase (PTK) Lyn is associated with Syk, respectively, through Src homology (SH) 2-phosphotyrosine interactions (7–9). Newly recruited PTKs are activated by transphosphorylation of tyrosine residues in their activation loops *Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA and by conformational changes in the case of Syk (10, 11). Active 92121; †Department of Microbiology and Molecular Genetics, University of Califor- nia, Los Angeles, CA 90095; ‡Department of Pediatrics, University of California, Los Lyn and Syk phosphorylate themselves and other protein sub- Angeles, CA 90095; §National Research Institute of Vegetables, Ornamental Plants strates such as phospholipase C (PLC)-␥1 and -␥2 (12–14). Acti- and Tea, Kanaya, Shizuoka, Japan; ¶Department of Laboratory Medicine, University ʈ vation of PLC leads to the generation of two second messengers, of California, San Francisco, CA 94143; and Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095 inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 mobilizes 2ϩ Received for publication November 11, 1999. Accepted for publication May 9, 2000. Ca from intracellular storage sites, and diacylglycerol together 2ϩ The costs of publication of this article were defrayed in part by the payment of page with Ca activates protein kinase C (PKC) (reviewed in Ref. 15). ϩ charges. This article must therefore be hereby marked advertisement in accordance Both Ca2 and PKC appear to be required for optimal mast cell with 18 U.S.C. Section 1734 solely to indicate this fact. degranulation (16). 1 This work was supported in part by National Institutes of Health Grant AI42244, Bruton’s tyrosine kinase (Btk) is a member of the Tec family of AI33617, and AI38348 (to T.K.). A.B.S. is a Special Fellow of the Leukemia Society of America. D.J.R. is a recipient of a McDonnell Scholar Award and is supported in kinases characterized by N-terminal pleckstrin homology (PH) and part by the facilities of the Jonsson Comprehensive Cancer Center, University of Tec homology domains (17–19). btk mutations affect B cell de- California Los Angeles. O.N.W. is an Investigator of the Howard Hughes Medical velopment and result in X-linked agammaglobulinemia in humans Institute. This article is Publication No. 286 from the La Jolla Institute for Allergy and Immunology. (17, 18) and X-linked immunodeficiency (xid) in mice (20, 21). 2 Address correspondence and reprint requests to Dr. Toshiaki Kawakami, La Jolla Btk is also highly expressed in mast cells and is required for cy- Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA tokine production in response to Fc⑀RI cross-linking (22). Unlike 92121. E-mail address: [email protected] B cells, mast cell development does not require Btk. This suggests 3 ⑀ The abbreviations used are: Fc RI, high-affinity IgE receptor; Btk, Bruton’s tyrosine redundant roles for Btk and other Tec family kinases, such as kinase; DNP, dinitrophenyl; HSA, human serum albumin; IP3, inositol 1,4,5-trisphos- phate; MAP, mitogen-activated protein; PKC, protein kinase C; PLC, phospholipase Itk/Emt, in some mast cell signaling pathways. Mechanistically, C; PTK, protein-tyrosine kinase; SH, Src homology; ITAM, immunoreceptor ty- btk mutations result in the loss of extracellular Ca2ϩ influx and the rosine-based activation motif; PH, pleckstrin homology; BCR, B cell receptor; PI3-K, 2ϩ phosphatidylinositol 3-kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular sig- sustained phase of Ca response in B cell receptor (BCR)-stim- nal-related kinase. ulated B cells (23, 24). This defective response may be due in part Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 1211 to reduced PLC-␥ activation. Downstream of these early activation Biolabs (Boston, MA). Pansorbin was purchased from Calbiochem (La events in mast cells and B cells, Btk mediates the activation of Jolla, CA). Other chemicals of highest grade were obtained from Sigma JNK1, JNK2, and, to a lesser extent, p38 (25). JNK regulates c-Jun (St. Louis, MO), unless otherwise mentioned. and other transcription factors that induce the transcription of Cells TNF-␣, IL-2, and other cytokine genes (26), accounting for the btkϪ/Ϫ and lynϪ/Ϫ mice, each on a mixed C57BL/6 ϫ 129/Sv genetic reduced cytokine production in Btk-deficient mast cells. ϩ/Ϫ ϩ/Ϫ background, were mated to generate btk lyn F1 progeny. These F1 Activation of Btk in BCR and Fc⑀RI signaling systems requires mice were mated to obtain wild-type, btkϪ/Ϫ, lynϪ/Ϫ, and btkϪ/ϪlynϪ/Ϫ both phosphatidylinositol 3-kinase (PI3-K) and Src family kinases mice (36). Genotyping was done by Southern blotting or PCR analysis of (27, 28). The product of PI3-K, phosphatidylinositol 3,4,5- mouse tail-derived DNAs. Mast cells were cultured as described previously trisphosphate, is believed to recruit Btk to the plasma membrane (44). Briefly, bone marrow cells derived from the femur of the 6- to 10- wk-old mice were cultured in RPMI 1640 medium supplemented with 10% (29), where it is phosphorylated on tyrosine 551 in its activation FCS, 100 ␮M nonessential amino acids, 50 ␮M 2-ME, and 8% conditioned loop by activated Lyn molecules (27). Btk phosphorylated on ty- medium of IL-3 gene-transfected cells (bone marrow-derived mast cell rosine 551 is enzymatically active and autophosphorylates tyrosine medium).