Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction

Leptin exerts its weight-reducing effects by binding to its receptor and ac tivating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an ap proach was developed in which bacterially expressed phosphorylated fragment s of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of t he cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosin es of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin tre atment. Affinity chromatography using a tyrosine-phosphorylated fragment sp anning Tyr 985 of Ob-Rb was used to identify proteins that bind to this sit e. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was iso lated from bovine and mouse hypothalamus by using this method. After cotran sfection of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2 into 293T cells, leptin results in direct binding of SHP-2 to the phosphorylated Tyr 985, The boun d SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y-->F 985 receptor mutant is cotransfected, In the absence of SHP-2 phosphorylation, the level of JAK 2 phosphorylation was increased. Tyrosyl phosphorylation of the leptin rece ptor and signal transducer and activater of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation o f SHP-2 by the leptin receptor results in a decreased phosphorylation of JA K2 and may act to attenuate leptin signal transduction. The method used in this report can in principle be used to isolate additional components of th e leptin, or other, signal transduction pathway.