Regulation of Highly Cytokinergic IgE-Induced Mast Cell Adhesion by Src, Syk, Tec, and Protein Kinase C Family Kinases 1 Jiro Kitaura,* Koji Eto, † Tatsuya Kinoshita,* Yuko Kawakami,* Michael Leitges, ‡ Clifford A. Lowell, § and Toshiaki Kawakami 2 * Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to FcRI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive FcRI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through  1 and  7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C  as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C  plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to FcRI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion. The Journal of Immunology, 2005, 174: 4495– 4504. M ast cells are the major effector cell type in IgE-medi- ated immediate hypersensitivity. Traditionally, it is thought that mast cells bound to Ag-specific FcRI, so-called sensitized mast cells, encounter multivalent Ag, and then IgE-bound receptors are aggregated or cross-linked, leading to cell activation (1). Activated mast cells secrete preformed and newly synthesized proinflammatory mediators involved in inflammatory processes of allergy, such as histamine, proteases, lipids, cyto- kines, and chemokines. The mouse FcRI consists of an IgE-bind- ing  subunit, a signal-amplifying  subunit, and disulfide-bonded two molecules of signal-generating  subunit (2). FcRI aggrega- tion leads to the activation of  subunit-bound Lyn, a prominent Src family protein-tyrosine kinase (PTK) 3 in mast cells (3). Lyn phosphorylates the tyrosine residues in the ITAMs of the  and  subunits (4). Phosphorylated ITAMs of  and  subunits, respec- tively, recruit Lyn and Syk, a member of another PTK family with two tandemly arranged Src homology 2 domains, leading to the activation of these PTKs. Active Lyn and Syk phosphorylate nu- merous cellular target proteins, leading to the activation of multi- ple signaling pathways, including PI3K and phospholipase C (PLC)-. An alternative pathway involving Fyn (another Src fam- ily PTK) was shown to activate PI3K (5). PLC hydrolyzes phos- phatidylinositol 4,5-bisphosphate into two second messengers, i.e., diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). IP 3 triggers Ca 2+ release from intracellular stores and then Ca 2+ in- flux. Increased DAG and Ca 2+ lead to the activation of protein kinase C (PKC) (reviewed in Ref. 6). Both PKC and Ca 2+ are required for optimal degranulation (7). Cell-cell and cell-extracellular matrix (ECM) interactions me- diated by integrins play a critical role in cell proliferation, differ- entiation, survival, apoptosis, and function. Activation of leuko- cytes by agonists that bind to diverse classes of receptors triggers ligand recognition by integrins. This process is termed inside-out signaling. Activation of mast cells by FcRI aggregation and stem cell factor (SCF) induces adhesion to fibronectin (FN) (8) predom- inantly via integrin  5  1 (9 –11). Upon FcRI aggregation and SCF stimulation, FN-adherent cells exhibit stronger effector func- tions such as histamine release and cytokine production than non- adherent cells (10). Adherent cells exhibit more intense tyrosine phosphorylation of certain proteins, including focal adhesion ki- nase p125 FAK , than nonadherent cells (12, 13). FcRI- and SCF- induced mast cell adhesion involves several signaling pathways: PI3K and PLC- are required for adhesion induced by both stimuli (14). Lyn and Ca 2+ influx seem to be required for FcRI aggre- gation- but not SCF-induced adhesion, and PKC may also be in- volved in FcRI aggregation- but not SCF-induced adhesion (11). We and others (15, 16) recently demonstrated that monomeric IgE can promote mast cell survival. This observation, together with earlier studies showing that IgE in the absence of cross-link- ing reagents (such as Ag or anti-IgE) can increase the surface expression of FcRI (17–19), has transformed the traditional view *Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; † Division of Vascular Biology, Department of Cell Biology, Scripps Re- search Institute, La Jolla, CA 92037; ‡ Max Planck Institute for Experimental Endo- crinology, Hannover, Germany; and § Department of Laboratory Medicine, University of California, San Francisco, CA 94143-0134 Received for publication November 10, 2004. Accepted for publication January 11, 2005. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by Public Health Service Grants AI50209 and AI/ GM38348 from the National Institutes of Health (to T.K.). Part of this study was performed during a tenure of fellowship from the American Heart Association (to K.E.). 2 To whom all correspondence should be addressed to Dr. Toshiaki Kawakami, Di- vision of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: toshi@liai.org 3 Abbreviations used in this paper: PTK, protein-tyrosine kinase; PLC, phospholipase C; DAG, diacylglycerol; IP 3 , inositol 1,4,5-trisphosphate; ECM, extracellular matrix; PKC, protein kinase C; SCF, stem cell factor; FN, fibronectin; HC, highly cytokin- ergic; PC, poorly cytokinergic; TNP, trinitrophenyl; BMMC, bone marrow-derived mast cell. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00