The long-term aims of this project are to understand the control of the production and function of white blood cells (particularly granulocytes and macrophages) at both the cellular and molecular level in order to devise improved therapies for the treatment of blood disorders and leukemia. Preliminary results using site-directed mutants of the cytoplasmic domain of the GM-CSF receptor beta-chain have suggested that the multiple actions of GM-CSF have their origins from different cytoplasmic modules within the receptor that initiate separate pathways leading to cell survival, proliferation, differentiation or clonal suppression of proliferation. Sin each of these pathways can be subverted in the generation of leukemias and since one pathway may be utilized by several different cytokines, the most selective therapeutics of the future will target individual cytoplasmic signaling modules. The major aims of the current proposal are to use two cytokine/receptor systems (GM-CSF and LIF) to unequivocally map specific receptor cytoplasmic domains and biochemical pathways to particular biological responses. A panel of receptor truncation, tyrosine and chimeri mutants will be expressed in a series of cell lines in which survival, macrophage differentiation, proliferation and clonal suppression can be measured. This work will be extended to in vivo studies with normal hemopoietic cells by transient transfection or long-term reconstitution of bone marrow cells from mice engineered to lack a functional GM-CSF receptor beta-chain gene and so can be forced to express only the mutated receptors. A molecular genetic approach will be used to identify the functionally necessary biochemical components of the LIF-induced differentiation pathway. LIF unresponsive M1 myeloid leukemic cells will be generated using selectable genetic elements and classified according to whether they are dominant or recessive mutants and to which complementation group they belong by cell fusion. LIF-responsiveness will then be rescued, again using selectable genetic elements, by transfection o infection with cDNA expression libraries and the critical mutated or delete proteins thus identifies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA022556-21
Application #
2894490
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Mufson, R Allan
Project Start
1978-03-01
Project End
2000-07-31
Budget Start
1999-04-01
Budget End
2000-07-31
Support Year
21
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Walter and Eliza Hall Institute Medical Research
Department
Type
DUNS #
City
Victoria
State
Country
Australia
Zip Code
VIC, -3052
Nicola, Nicos A; Babon, Jeffrey J (2015) Leukemia inhibitory factor (LIF). Cytokine Growth Factor Rev 26:533-44
Babon, Jeffrey J; Varghese, Leila N; Nicola, Nicos A (2014) Inhibition of IL-6 family cytokines by SOCS3. Semin Immunol 26:13-9
Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M et al. (2014) The molecular regulation of Janus kinase (JAK) activation. Biochem J 462:1-13
Kershaw, Nadia J; Laktyushin, Artem; Nicola, Nicos A et al. (2014) Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates. Growth Factors 32:1-10
Kedzierski, Lukasz; Linossi, Edmond M; Kolesnik, Tatiana B et al. (2014) Suppressor of cytokine signaling 4 (SOCS4) protects against severe cytokine storm and enhances viral clearance during influenza infection. PLoS Pathog 10:e1004134
Varghese, Leila N; Ungureanu, Daniela; Liau, Nicholas P D et al. (2014) Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses. Biochem J 458:395-405
Babon, Jeffrey J (2013) Quantitative analysis of JAK binding using isothermal titration calorimetry and surface plasmon resonance. Methods Mol Biol 967:57-67
Kershaw, Nadia J; Murphy, James M; Liau, Nicholas P D et al. (2013) SOCS3 binds specific receptor-JAK complexes to control cytokine signaling by direct kinase inhibition. Nat Struct Mol Biol 20:469-76
Kolesnik, Tatiana B; Nicholson, Sandra E (2013) Analysis of Suppressor of Cytokine Signalling (SOCS) gene expression by real-time quantitative PCR. Methods Mol Biol 967:235-48
Babon, Jeffrey J; Laktyushin, Artem; Kershaw, Nadia J (2013) In vitro ubiquitination of cytokine signaling components. Methods Mol Biol 967:261-71

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