JAK-3 AND MYELOID CELL DIFFERENTIATION

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DESCRIPTION: (Adapted from investigator's abstract) The Janus Kinases (JAK) are a family of protein tyrosine kinases (which include JAK1, JAK2, and TYK2) that play a pivotal role in the signal transduction process mediated by cytokines and interferons. JAK kinases differ from other cytoplasmic kinases by their lack of SH2 and SH3 domains and by the presence of two kinase domains. It is becoming increasingly evident that certain cytokines such as Granulocyte Colony Stimulating Factor (G-CSF) can transmit signals for both cellular proliferation and differentiation. It is at present unclear whether both of these signals are transmitted by the same JAK kinase or whether an entire family of such kinases are involved in this process. It can be hypothesized that in an immature myeloid cell, a cytokine such as G-CSF may induce the phosphorylation of a JAK kinase, and with continued exposure to cytokines, expression of new JAK kinases are induced which transmit a new set of signals resulting in the induction of genes associated with cell growth arrest and terminal differentiation. The principal investigator has recently identified a new member of the JAK kinase family, named JAK3. JAK3 is selectively expressed in myeloid and lymphoid cells and appears to be a primary response gene induced by G-CSF and IL6. JAK3 is expressed at very low levels in immature hematopoietic cells and is up-regulated with terminal differentiation. In preliminary studies, the applicant has shown that forced expression of JAK3 in a murine myeloid cell line (32Dcl3) accelerates the G-CSF-induced differentiation program. Thus, this investigator hypothesizes that while the JAK1 and JAK2 kinases bind to the G-CSF receptor and play a critical role in delivering the G-CSF mitogenic signal, JAK3 is associated with providing signals that promote terminal differentiation. The experiments proposed in this application are aimed at understanding the role of JAK-3 in the terminal differentiation process of myeloid cells along the granulocytic and monocytic pathways. The aims include: 1) to extend previous studies with 32Dcl3 cells (capable of differentiating only to granulocytes) to the M1 cell line (capable of monocytic differentiation in response to IL6); since JAK kinases are known to exert their biological effects via phosphorylation of STATS, it will be determined whether JAK3 interacts with and phosphorylates any STATs expressed in 32Dc13 and M1 cells exposed to G-CSF or and IL-6 respectively; 2) to determine if JAK3 is expressed in normal murine bone marrow cells in response to IL6, G-CSF, and GMCSF, and to determine whether induction of JAK3 is due to new transcription or post-transcriptional mechanisms; 3) to determine how myeloid cell differentiation is affected when JAK-3 expression is inhibited using anti-sense oligonucleotides; in addition, preliminary results suggest that the v-abl, v-myb, EGR-1 and HOX-2.4 genes inhibit the ability of G-CSF to induce granulocytic differentiation of the 32Dc13 cell line due to inhibition of the induction of JAK-3 expression. Using these model systems, it will be determined if constitutive expression of JAK3 using exogenous promoters will rescue the 32D/v-abl, 32D/v-myb, 32D/EGR and 32D/Hox2.4 cell lines from the block to granulocytic differentiation following stimulation with G-CSF; 4) to determine how JAK3 promotes terminal differentiation through cell cycle arrest; 5) to carry out detailed analysis of the promoter/enhancer region of JAK3 to examine sequence elements that play a crucial role in cytokine responsiveness; and 6) to identify genes regulated by the JAK3 pathway.