Identification of a binding partner and regulatory site that inhibit the function of the cytokine receptor -associated tyrosine kinase JAK2.

Abstract

Activation of the tyrosine kinase JAK2 is an essential step in signaling by growth hormone (GH) and other ligands of the cytokine family of receptors. To gain insight into how JAK2 contributes to cellular responses to ligand, I looked for novel JAK2 regulatory sites and binding proteins. This work identifies phosphorylated serine 523 (Ser 523) in JAK2 as a negative regulatory site of JAK2 kinase activity. Ser 523 is rapidly but transiently phosphorylated in response to GH. MEK1 inhibitor suppresses GH-dependent phosphorylation of JAK2 at Ser 523, suggesting that ERKs 1 and/or 2 phosphorylate Ser 523 in response to GH. Other activators of ERKs, phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor, stimulate phosphorylation of Ser 523. Mutation of Ser 523 to alanine enhances GH-dependent tyrosyl phosphorylation of JAK2 and STAT5, suggesting that phosphorylation of Ser 523 inhibits JAK2 kinase activity. Thus, phosphorylation of Ser 523 by ERKs 1/2 in JAK2 may act in a negative feedback manner to dampen the initial activation of JAK2 in response to GH and provide a mechanism by which prior exposure to ERK activating ligands might dampen the cellular response to GH. I also used the yeast two-hybrid system to identify steroid sensitive gene 1 (SSG1) as a novel JAK2 binding protein. I provide evidence that SSG1 binds preferentially to the active form of JAK2, is phosphorylated by JAK2, and localizes to the nucleus, perinuclear region and the plasma membrane. SSG1 inhibits GH-induced expression of the <italic>c-fos</italic> gene, suggesting that SSG1 is a signaling protein involved in GH regulation of gene transcription. Furthermore, I identified an SRPX5 domain in SSG1 as a novel JAK2 interaction domain. This domain is common to a newly emerging group of proteins that includes rat SSG1/CL2/DRO1, its mouse and human homolog upregulated in BRS-3 deficient mice (Urb), chicken ortholog Equarin, and SRPX/drs/ETX1 and SRPUL. The SRPX5 domain is both necessary and sufficient for binding to JAK2 in a phosphotyrosine dependent manner and may therefore be a novel protein-protein, possibly phosphotyrosine binding, interaction domain. Taken together, this work provides important insight into signaling mechanisms used by the cytokine receptor family by identifying a binding partner and regulatory site in JAK2, both of which inhibit its ability to activate cytokine signaling pathways.