Binding of SH2-B family members within a potential negative regulatory region maintains JAK2 in an active state

The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-B beta and APS regulate the activity of JAK2. We show that like SH2-B beta, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 1,: essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-BP increase JAK2 activity independent of their N-terminal dimerization domains. SH2-B beta-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-BP to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology I (JH1) and JH2 domains and possibly the FEM domain. Together, our data suggest that SH2-BP and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.