JAK2 is required for induction of the murine DUB-1 gene.

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the bc subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1‐luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1‐luc. A dominantnegative form of Raf-1 inhibited the induction of DUB-1‐luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5. Interleukin-3 (IL-3) is a glycoprotein hormone that regulates the growth and differentiation of hematopoietic progenitor cells (17). IL-3 activates a specific receptor (IL-3R) which is expressed on the surface of target cells. The IL-3R complex consists of an IL-3-specific a chain and a common b chain, bc, shared by the granulocyte-macrophage colony-stimulating factor receptor and the IL-5 receptor (20, 37). The IL-3R activates multiple signal transduction pathways, including the Ras/ Raf/mitogen-activated protein (MAP) kinase pathway and the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway, resulting in the induction of immediate-early genes. How these immediate-early genes couple receptor activation to the biochemical machinery of cell growth and cell cycle progression is poorly understood. Discrete domains of the cytoplasmic region of the bc subunit activate the Ras/Raf/MAP kinase pathway and the JAK/STAT pathway. The distal cytoplasmic domain of bc, for example, activates the Ras/Raf/MAP kinase pathway (33, 34). This region is also required for the induction of the immediate-early genes c-fos and c-jun (34). The membrane-proximal domain of bc, in contrast, is required for activation of the JAK/STAT pathway (45). The IL-3R, like other cytokine receptors, activates a combination of JAK and STAT proteins (7, 15). JAK kinases are cytoplasmic proteins that constitutively associate with the membrane-proximal region of cytokine receptors and become activated upon receptor-ligand interaction. STAT proteins are latent cytoplasmic transcription factors that are phosphorylated by JAK kinases. Seven STAT proteins are known at present. Phosphorylated STAT proteins form homo- and hetero-oligo