Binding of SH2-B Family Members within a Potential Negative Regulatory Region Maintains JAK2 in an Active State

Abstract

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-Bβ and APS regulate the activity of JAK2. We show that like SH2-Bβ, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is 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-Bβ increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bβ-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bβ 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 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bβ 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.

This work was supported by NIH grants DK34171 and DK54222 (to C.C.-S.). J.H.K. was supported by Predoctoral Fellowship of the Cellular and Molecular Approaches to Systems and Integrative Biology Training Grant T32-GM08322 and the University of Michigan Medical Scientist Training Program (NIH T32 GM07863). cDNA sequencing and synthesis of the phosphorylated and unphosphorylated peptides were supported by the Cellular and Molecular Biology Core and the Protein Core, respectively, of the Michigan Diabetes Research and Training Center (P60-DK20572), by the University of Michigan Multipurpose Arthritis Center (P60-AR20557), and by the University of Michigan Comprehensive Cancer Center (NIH P30 CA46592).

We thank Xiaqing Wang for support and assistance with DNA constructs, Lawrence S. Argetsinger for critical comments on the manuscript and help with the figures, and Barbara Hawkins for assistance with the manuscript.