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SH2 Domain-Containing Phosphatase-2 Protein-Tyrosine Phosphatase Promotes FcRI-Induced Activation of Fyn and Erk Pathways Leading to TNF Release from Bone Marrow-Derived Mast Cells¹

Victor A. McPherson,^* Namit Sharma,^* Stephanie Everingham,^* Julie Smith,^* Helen H. Zhu, Gen-Sheng Feng,² and Andrew W. B. Craig^3*

^*Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada; Burnham Institute for Medical Research, La Jolla, California 92037; and Division of Cancer Biology and Genetics, Queen’s Cancer Research Institute, Kingston, Ontario, Canada

Clustering of the high affinity IgE receptor (FcRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FcRI signaling involves rapid tyrosine phosphorylation of FcRI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in FcRI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-derived mast cells (BMMCs), we provide insights into SHP2 functions in the FcRI pathway. Although no overt defects in FcRI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for FcRI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IB kinase, and decreased TNF- release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of FcRI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF- release from mast cells.

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 work was supported by Canadian Institutes of Health Research (CIHR) operating Grant (MOP82882) (to A.W.B.C.); A.W.B.C. is supported by a CIHR New Investigator award.

² Current address: Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, CA 92093.

³ Address correspondence and reprint requests to Dr. Andrew Craig, Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada. E-mail address: ac15{at}queensu.ca

⁴ Abbreviations used in this paper: SFK, Src family kinase; PTK, protein-tyrosine kinase; LAT, linker for activation of T cells; NTAL, non-T cell activation linker; IKK, IB kinase; PTP, protein-tyrosine phosphatase; MAFA, mast cell function-associated Ag; SHP2, Src homology region 2 domain-containing phosphatase 2; EGFR, epidermal growth factor receptor; Csk, C-terminal Src kinase; 4OH-TM, 4-hydroxytamoxifen; BMMC, bone marrow-derived mast cell; SCF, stem cell factor; WT, wild type; KO, knockout; IB, immunoblot; KLB, kinase lysis buffer; SCL, soluble cell lysate; pAkt, phosphorylated Akt; EL, epilepsy prone; Gab2, Grb2-associated binder 2; Cbp/PAG, Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains; RasGAP, Ras GTPase-activating protein; CreER*, Cre recombinase-estrogen receptor fusion; PLC, phospholipase C-; SHP1, Src homology region 2 domain-containing phosphatase 1; DNP-HSA, dinitrophenyl-human serum albumin.