The mechanism by which the adaptor protein SH2-Bbeta enhances the activity of the cytokine receptor -associated tyrosine kinase JAK2.

The receptor associated tyrosine kinase, Janus Kinase 2 (JAK2), transduces signaling for the majority of known cytokine receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Two-dimensional phosphopeptide mapping and a phosphospecific antibody identified tyrosine 813 as a site of autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Phosphorylation of tyrosine 813 was shown to be required for the SH2 domain-containing adaptor protein SH2-Bbeta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is required for SH2-Bbeta to bind JAK3. Although expression of SH2-Bbeta with JAK2 increases the amount of active JAK2, expression of the SH2-B family member, APS, either increases or decreases active JAK2, depending on JAK2 levels. I tested several hypotheses concerning the mechanism by which SH2-Bbeta increases active JAK2, and examined the basis for the differential effects of APS and SH2-Bbeta on JAK2. Similar to SH2-Bbeta, APS binds JAK2 at multiple regions but its binding at phosphotyrosine 813 is essential for APS to enhance JAK2 activity. Neither APS nor SH2-Bbeta requires residues N-terminal to its SH2 domain to enhance JAK2 activity. Indeed, SH2-Bbeta requires only its SH2 domain to enhance both JAK2 activity and JAK2 multimerization. The minimum region of JAK2 required for SH2-Bbeta to increase JAK2 activity includes amino acids 807-1129, composed of the kinase domain and SH2-BP binding site. Further examination of amino acids surrounding tyrosine 813 revealed a regulatory region in JAK2, amino acids 809-811, that when mutated increases the amount of active JAK2. Together these data indicate that neither APS nor SH2-Bbeta require their N-terminal dimerization domains to increase active JAK2, and that SH2-Bbeta most likely does not serve to recruit an activator of JAK2. Rather, SH2-Bbeta either induces a conformational change involving residues 809-811 of JAK2, or competes with a JAK2 inhibitor that binds this region.PhDBiological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126474/2/3253322.pd