Regulation of protein kinase C␤Ibytwo protein-tyrosine kinases, Btk and Syk Yuko Kawakami*, Jiro Kitaura*, Stephen E. Hartman*, Clifford A. Lowell†, Reuben P. Siraganian‡, and Toshiaki Kawakami*§ *Division of Allergy, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121; †Department of Laboratory Medicine, University of California, San Francisco, CA 94143; and ‡Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research͞National Institutes of Health, Bethesda, MD 20892 Communicated by Kimishige Ishizaka, Kirin Brewery Co., Ltd., Tokyo, Japan, April 18, 2000 (received for review January 1, 2000) Two protein-tyrosine kinases, Bruton’s tyrosine kinase (Btk) and is shown to regulate the JNK pathway that leads to transcrip- Syk, and members of the protein kinase C (PKC) subfamily of tional activation of cytokine genes. serine͞threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE Materials and Methods receptors (Fc␧RI) in mast cells. The present study provides genetic, Cell Culture and Stimulation. Bone marrow cells derived from biochemical, and pharmacological evidence that, on Fc␧RI stimu- wild-type (wt), btk knockout (btkϪ) (21), and lyn knockout lation, Syk regulates Btk, and Btk selectively regulates the mem- (lynϪ) (22) mice were cultured in IL-3-containing medium for brane translocation and enzymatic activity of PKC␤I among the 4–6 wk to generate Ͼ95% pure populations of mast cells. Cells conventional PKC isoforms (␣, ␤I, and ␤II) expressed in mast cells. were sensitized overnight with anti-dinitrophenyl (DNP) IgE Syk͞Btk-mediated PKC␤I regulation is involved in transcriptional monoclonal antibody and stimulated with antigen, DNP-human activation of the IL-2 and tumor necrosis factor ␣ genes through the serum albumin conjugates. Retroviral transfection of btk- mast JNK pathway induced by Fc␧RI stimulation. Accordingly, Fc␧RI- cells was done as described (23). Wt, Syk-deficient (sykϪ) induced production of these cytokines is inhibited by specific variant, and syk cDNA-transfected sykϪ RBL-2H3 cells (13) inhibitors of Btk and Syk, as well as broad-specificity inhibitors of were similarly stimulated with IgE and antigen. PKC and a selective inhibitor of PKC␤. Specific regulation of PKC␤I IMMUNOLOGY by Btk is consistent with the selective association of Btk with Mutagenesis and Transfection. Two-step PCR mutagenesis was PKC␤I. Components of this signaling pathway may represent an performed to generate mutant rat PKC cDNAs. COS-7 and bone attractive set of potential targets of pharmaceutical interference marrow-derived mast cells were electroporated with plasmid for the treatment of allergic and other immunologic diseases. constructs. Luciferase reporter assays were performed as de- scribed (24). tk (Bruton’s tyrosine kinase) and Syk are protein-tyrosine Immunoblotting Analysis and Antibodies. Subcellular fractionation kinases that play crucial roles in B cell and mast cell B was performed as described (25). Otherwise, cells were lysed in activation (1–3). Mutations in the btk gene lead to X-linked 1% Nonidet P-40-containing lysis buffer (20 mM Tris⅐HCl, pH agammaglobulinemia in humans (4, 5) and X-linked immuno- 8.0͞0.15 M NaCl͞1 mM EDTA͞1 mM sodium orthovanadate͞1 deficiency (xid) in mice (6, 7). Btk mutations also result in mM phenylmethylsulfonyl fluoride͞10 ␮g/ml aprotinin͞10 defective cytokine production in the affected mast cells on ␮g/ml leupeptin͞25 ␮M p-nitrophenyl pЈ-guanidinobenzoate͞1 Fc␧RI stimulation (8). Syk gene inactivation results in profound ␮M pepstatin͞0.1% sodium azide). Proteins in cleared cell hematopoietic defects, including B cell development (9, 10). lysates or subcellular fractions were either immunoprecipitated Loss of Syk expression ablates B cell receptor (BCR)- or before or directly analyzed by SDS͞PAGE followed by immu- Fc␧RI-mediated cell activation (11–13). Engagement of BCR ␧ noblotting. Antibodies used for immunoprecipitation and blot- and Fc RI elicits the enzymatic activation of receptor-bound Src ting were anti-Btk (M138), anti-PKC␣ (C-20), anti-PKC␤I (C- family protein-tyrosine kinases, such as Lyn. These kinases are 16), anti-PKC␤II (C-18), anti-PKC␦ (C-20), anti-PKC␧ (C-15), believed to phosphorylate tyrosine residues in the immunore- anti-PKC␩ (C-15), anti-PKC␪ (C-18), anti-PKC␨ (C-20), anti- ceptor tyrosine-based activation motifs (ITAMs) in signaling Lyn (44), anti-Syk (C-20), anti-JNK1 (C-17), anti-MEKK1 (C- subunits of receptor. Tyrosine-phosphorylated ITAMs recruit 22), anti-MEK4 (K-18) antibodies (all from Santa Cruz Biotech- Src family and Syk kinases through Src homology 2 (SH2) nology) unless otherwise mentioned, and proteins reactive with domain-phosphotyrosine interactions and activate these kinases. primary antibody were visualized with a horseradish peroxidase- Both btk and syk mutations impair the Ca2ϩ response on BCR ␧ conjugated secondary antibody and enhanced chemilumines- or Fc RI engagement, because of defective activation of cence reagents (NEN Life Science Products). phospholipase C (PLC)-␥ (11–17). PLC-␥ hydrolyzes phospha- tidylinositol 4,5-bisphosphate into diacylglycerol and inositol Kinase Assays. PKC assays were performed by two methods. Cells 1,4,5-trisphosphate (IP3). Diacylglycerol activates several were lysed and immunoprecipitated with anti-PKC␣, PKC␤I, or 2ϩ protein kinase C (PKC) isoforms, and IP3 recruits Ca from PKC␤II antibodies (Santa Cruz Biotechnology). In a protocol, intracellular storage sites. PKC is a family of serine͞threonine kinases that play crucial roles in a plethora of biological func- tions, such as proliferation, differentiation, development, and Abbreviations: BCR, B cell receptor; Btk, Bruton’s tyrosine kinase; DNP, dinitrophenyl; PKC, ␧ more specialized cellular functions (18–20). Based on cofactor protein kinase C; Fc RI, high-affinity IgE receptors; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; TNF, tumor necrosis factor; wt, wild type; xid, X-linked immunodeficiency; requirements and structure, PKC family members are divided PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; GST, glutathione S-transferase. into the Ca2ϩ͞diacylglycerol-regulated conventional isoforms §To whom reprint requests should be addressed. E-mail: [email protected]. (cPKC: ␣, ␤I, ␤II, and ␥), the Ca2ϩ-independent but diacylglyc- ␦ ␧ ␩ ␪ ␮ The publication costs of this article were defrayed in part by page charge payment. This erol-regulated novel isoforms (nPKC: , , , , and ), and the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 2ϩ Ca ͞diacylglycerol-independent atypical isoforms (aPKC: ␨ §1734 solely to indicate this fact. ␫͞␭ and ). In the present study, we provide evidence that Syk Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.120175097. regulates Btk and that Btk regulates PKC␤I activation. PKC␤I Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.120175097 PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7423–7428 Downloaded by guest on October 2, 2021 the immunoprecipitates were subjected to autophosphorylation. Fifty-microliter reactions in kinase buffer (20 mM Tris, pH ͞ ͞ ␮ ␮ 7.4 10 mM MgCl2 10 M ATP) in the presence of 10 Ci [␥-32P]ATP were analyzed by SDS͞PAGE followed by electro- blotting and autoradiography. In another protocol, PKC␤I im- munoprecipitates were incubated in the same buffer with a peptide substrate based on myelin basic protein (EKRPSQRS- KYL) plus 10 ␮Ci [␥-32P]ATP and cofactors (Ca2ϩ, diacylglyc- erol, and phosphatidylserine). Radioactivity incorporated into phosphorylated peptides that were recovered with SpinZyme separation units (Pierce) was counted. Btk activity was measured by autophosphorylation reactions of immune complexes precip- itated by anti-Btk (M138). For JNK assays, anti-HA (12CA5; Roche Molecular Biochemicals) immunoprecipitates were incu- bated with 3 ␮g GST-c-Jun (1–79) in 15-min reactions at 30°C in 20 mM Hepes, pH 7.4, 10 mM MgCl2,22mMDTT,20mM ␤ ␮ ␮ ␮ -glycerophosphate, 50 MNa3VO4,20 M ATP, and 10 Ci [␥-32P]ATP. Reactions were analyzed by SDS͞PAGE, blotting, and autoradiography. Results Btk Regulates Membrane Translocation and Activation of PKC␤I. In light of the recent studies that Btk regulates the sustained increase of intracellular Ca2ϩ levels induced by BCR stimulation ␧ (16, 17), we compared IP3 production in Fc RI-stimulated bone marrow-derived mast cells from wt and btkϪ mice. As expected, Ϫ btk mast cells produced 40% less amounts of IP3 at their peak (30 s after Fc␧RI stimulation) compared with wt cells (unpub- lished observations). This data and the defective calcium re- sponse in receptor-engaged Btk-deficient B and mast cells (14–17) suggest that the activity of cPKC isoforms may be affected by btk mutation. Therefore, we examined the subcellular locations and activities of these PKC isoforms in mast cells, except for the ␥ isoform that is not expressed in mast cells. Mast Fig. 1. Btk is required for the membrane translocation and activation of cells were fractionated into the cytosolic and particulate com- PKC␤I. (A) Mast cells from wt or btkϪ mice were passively sensitized with partments. As reported previously (26), a time-dependent trans- anti-DNP IgE antibody, and stimulated by an antigen, 100 ng͞ml DNP conju- location of cPKC isoforms from the cytosol to the particulate gates of human serum albumin,