The Kinase Domain of Jak2 Mediates Induction of Bcl-2 and Delays Cell Death in Hematopoietic Cells

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 stimulate DNA synthesis and proliferation and inhibit apoptosis in hematopoietic cells. Multiple signal pathways are activated by binding of these ligands to their receptors, which share a common ␤ subunit. Janus protein kinase 2 (Jak2) binds to the membrane proximal domain of the ␤ chain and is phosphorylated on receptor ligation. To explore the role of Jak2 in the regulation of specific signal transduction pathways, we constructed fusion proteins with a CD16 external domain, a CD7 transmembrane region, and a Jak2 cytoplasmic domain. This cytoplasmic domain consisted either of wild type Jak2 (CD16/Jak2-W) or Jak2 mutations with deletions of (a) the amino terminus (CD16/Jak2-N), (b) kinase-like domain (CD16/Jak2-B), (c) kinase domain (CD16/Jak2-C), or (d) amino-terminal and kinase-like domains, leaving the kinase domain (CD16/Jak-K) intact. In contrast to the CD16/Jak2-W fusion protein, which requires crosslinking for activation, CD16/Jak2-N, CD16/Jak2-B, and CD16/Jak2-K were constitutively phosphorylated, and they stimulated Shc phosphorylation and increased binding of STAT to DNA in Ba/F3 cells. Cell lines derived from IL-3-dependent Ba/F3 cells stably transfected with CD16/Jak2-W, CD16/Jak2-N, or CD16/Jak2-B mammalian expression vectors died at a rate similar to that of the parental cells on IL-3 deprivation. In contrast, CD16/ Jak2-K cell lines exhibited increased expression of bcl-2 and pim-1 mRNA and maintained their viability when compared with control cell lines. Thus, activation of tyrosine phosphorylation by creating a CD16/Jak2-K fusion is sufficient to activate pathways that prevent cell death.

Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

“…Factor depletion-induced apoptosis occurred even in the presence of coumermycin implied that GyrB-Jak2 activation is insufficient to prevent apoptosis. In an apparent contradiction with our present finding, a previous report (Sakai and Kraft, 1997) demonstrated that activation of CD16 -Jak2-kinase domain fusion is sufficient to prevent cells from apoptosis and showed constitutive phosphorylation of Shc, which also has been implicated in transducing signals to the Ras-MAP kinase pathway. Our series of analysis of antiapoptosis activity of hGM-CSF using various ␤c mutants revealed that Jak2 activation through the box 1 region is essential, and that activation of either a tyrosine kinase inhibitor genisteinsensitive pathway or the MAP kinase cascade is sufficient to sustain cell viability (Liu, Itoh, and Watanabe unpublished data).…”

Janus Kinase 2, an Early Target of α7 Nicotinic Acetylcholine Receptor-mediated Neuroprotection against Aβ-(1–42) Amyloid

“…Anti-apoptotic signals transduced via JAK2 have also been reported. For example, in hematopoietic cells, the kinase domain of JAK2 mediates the induction of Bcl-2 and inhibits cell death (19), and treatment with the JAK2 inhibitor AG-490 reduces the phosphorylation of PI-3-K (20) and STAT3 resulting in an increase in caspase-3 activity and Bax protein in acute myocardial infarction (21). In addition, activation of neuronal erythropoietin receptors prevents apoptosis by triggering cross-talk between the signaling pathways of JAK2 and the nuclear factor-B (22).…”

Regulation of Proliferation, Differentiation and Survival by the IL-3/IL-5/GM-CSF Receptor Family

“…Jak2 activation by these cytokines seems to be involved in the induction of Bcl2 [137]. Interestingly, overboth the Ras-ERK and the PI3K-PKB pathways can have roles in cellular survival, depending on the cell type studied, ability of specific inhibitors of the MAPK and PI3K pathways will undoubtedly be of great help in determining the a number of questions remain unanswered.…”