Growth hormone (GH) treatment of cells promotes activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2 and JAK2 molecules with deletions of the amino terminus (JAK2ATD), carboxyl terminus (JAK2CTD), or kinase-like domain (JAK2PKD) were each transiently coexpressed in COS-7 cells with the rabbit GHR. The following responses were assayed: GH-induced transactivation of a luciferase reporter governed by a c-fos enhancer element; GH-induced shift in the molecular mass of a cotransfected epitope-tagged extracellular signal-regulated kinase molecule; and GH-induced antiphosphotyrosine immunoprecipitability of the transfected JAK2 form. In each assay, WTJAK2 and JAK2PKD allowed GH-induced signaling, whereas JAK2ATD and JAK2CTD did not. Anti-GHR serum coimmunoprecipitated WTJAK2, JAK2PKD, and JAK2CTD, but not JAK2ATD. Finally, a chimera in which the JAK2 kinase domain replaced the GHR cytoplasmic domain signaled GH-induced transactivation. We conclude: 1) kinase-like domain deletion eliminates neither physical nor functional interaction between JAK2 and the GHR; 2) kinase domain deletion eliminates functional but not physical coupling of JAK2 to the GHR; 3) interaction with the GHR appears dependent on the NH2-terminal one-fifth of JAK2; and 4) a GH-responsive signaling unit can include as little as the GHR external and transmembrane domains and the JAK2 kinase domain.
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.
IntroductionInterferon ␣ (IFN-␣) is an important therapeutic cytokine that exerts antitumor activity in a variety of tumor cells. 1,2 Chronic myelogenous leukemia (CML) is one of the hematologic malignancies that responds well to IFN-␣ therapy. [3][4][5] However, the effect of the therapy is limited because of the development of resistance to IFN-␣, which has often been observed in patients with CML in the late chronic phase, accelerated phase, or blastic phase. 5 Although efforts to understand the molecular basis of the resistance to IFN-␣ have been made, the mechanism is still unknown.Interferon ␣ exerts its biologic actions by binding to the high-affinity cell surface receptor. Receptor-associated Janus family tyrosine kinases Tyk2 and Jak1 are activated on stimulation by IFN-␣, followed by tyrosine phosphorylation of critical tyrosine residues of the cytoplasmic domain of the receptors by Jaks. 6 This allows receptor recruitment and Jak-mediated tyrosine phosphorylation of signal transducer and activator of transcription (STAT) molecules. When STAT1 and STAT2 become tyrosine phosphorylated they bind to each other and, in combination with p48, form a complex called IFN-stimulated gene factor-3 (ISGF3). After translocation into the cell nucleus, this complex binds to the conserved IFN-stimulated responsive element (ISRE) sequence within the promoter of IFN-responsive genes and initiates transcription of these genes. 7 The suppressor of cytokine signaling (SOCS) proteins, 8 also known as STAT-induced STAT inhibitor (SSI) 9 or cytokineinducible src homology (SH)2 domain-containing protein (CIS), 10 are a family of negative regulators of cytokine signaling that are characterized by a central SH2 domain and a C-terminal SOCSbox. 11 Of the family members, SOCS1 and SOCS3 are the most potent inhibitors of cytokine-induced signals. Forced expression of SOCS1 or SOCS3 down-regulates a variety of cytokine signal pathways including Previously, we established a new human CML cell line, KT-1, from the peripheral blood of a patient with CML blast crisis. 13 Although most CML cell lines are resistant to IFN-␣, this cell line is sensitive to the antiproliferative and apoptosis-inducing effects of IFN-␣. Subsequently, we established several sublines of the KT-1 cell line. 14,15 These sublines exhibit significant variation in responsiveness to IFN-␣. One subline, KT-1/A3, is the most sensitive cell line against IFN-␣ treatment. One of the IFN-␣-resistant sublines, KT-1/B7, was isolated by subcloning of KT-1 cells without any selection by IFN-␣ treatment. 14 Another IFN-␣-resistant cell line, KT-1/A3R, was isolated by culturing KT-1/A3 cells with increasing concentrations of IFN-␣. 15 In both of these IFN-␣-resistant sublines, IFN-␣-induced activation of ISGF3 was reduced in comparison with KT-1/A3 cells. In the KT-1/B7 subline, the level of STAT2 protein (one of the ISGF3 components) was reduced and this reduction was responsible for the reduced ISGF3 activation by IFN-␣. In the KT-1/A3R subline, the ISGF3 components were unc...
The t(1;21)(p36;q22) is a recurrent chromosome abnormality associated with therapy-related acute myeloid leukemia (AML). Although involvement of RUNX1 has been detected by fluorescence in situ hybridization analysis, the partner gene has not been reported previously. We identified a novel RUNX1 partner gene, MDS1/EVI1-like-gene 1 (PRDM16), in an AML patient with t(1;21). Alternative splicing of the fusion gene generates five different fusion transcripts. In two of them, the PRDM16 reading frame is maintained in the fusion with RUNX1, suggesting that the RUNX1-PRDM16 gene fusion results in the production of a protein that is highly homologous to the RUNX1-MDS1/EVI1 chimeric protein. It is suggested that PRDM16 and MDS1/EVI1 share a common molecular mechanism for the leukemogenesis of RUNX1-associated leukemia. Characterization of the RUNX1-PRDM16 fusion protein and comparison with the RUNX1-MDS1/EVI1 protein will facilitate the understanding of the mechanisms underlying RUNX1-associated leukemia.
A t(4;11)(q21;q23) has been described in 50-70% of cases of infant acute lymphoblastic leukemia and, less frequently, in cases of pediatric and adult acute lymphoblastic leukemia and acute myeloid leukemia (AML). In t(4;11)(q21;q23) leukemias, the AF4 gene has been cloned as a fusion partner of the MLL gene. A human myeloid leukemia cell line, chronic neutrophilic leukemia (CNL)BC1, was established from a peripheral blood specimen of a patient with CNL in leukemic transformation. As with the original leukemia cells, the established line had a t(4;11)(q21;q23). We showed that the MLL gene on 11q23 was fused to the FLJ10849 gene on 4q21. The protein encoded by FLJ10849 belongs to the septin family, sharing highest homology with human SEPT6, which is one of the fusion partners of MLL in t(X;11)(q13;q23) AML. Our results suggest that FLJ10849 might define a new septin family particularly involved in the pathogenesis of 11q23-associated leukemia. The established cell line, CNLBC1, could provide a useful model for analyzing the pathogenesis of MLL-septin leukemias and chronic neutrophilic leukemia.
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