The role of βc subunit phosphorylation in the functioning of the GM-CSF/IL-3/IL-5 receptors.

The cytokines GM-CSF, IL-3 and IL-5 are central regulators of haemopoietic cell functions and are pivotal in the regulation of haemopoiesis and inflammatory responses of myeloid cells. In particular, these cytokines have been shown to perform essential functions in host defence against foreign pathogens through their ability to regulate innate immune responses in myeloid cells. As key regulators of such important processes, these cytokines play an important role in human inflammatory pathologies such as rheumatoid arthritis, asthma, multiple sclerosis and psoriasis as well as a number of leukemias such as JML and CMML. GM-CSF, IL-3 and IL-5 signal through receptors containing α subunits specific to each cytokine and a common β subunit (βc). Cytokine stimulation leads to tyrosine phosphorylation of the βc and promotes specific responses such as proliferation, survival and activation of haemopoietic cells. Mouse knockout studies identified a key function of these cytokines in the activation of effector functions of myeloid cells, including production of reactive oxygen species (ROS) and phagocytosis. These earlier studies provide a link between cytokine signalling and inflammation, but the molecular mechanisms by which βc activation regulates effector cell functions, and the receptor motifs involved, are unknown. The aim of this thesis was to address two broad questions with regard to βc signalling: (1) Does βc regulate specific cellular responses by phosphotyrosine-independent mechanisms? (2) What are the molecular mechanisms by which βc initiates signalling to promote specific biological responses such as activation of effector cell functions? To address the first question, we have focussed on Serine 585, a potential 14-3-3 binding site which lies in the cytoplasmic potion of huβc. Out results show that the mutation huβc S585G disrupted the interaction of 14-3-3ζ with βc, whilst not affecting receptor tyrosine phosphorylation. Both mouse and human βc were shown to interact with 14-3-3 proteins, indicating that this interaction is conserved between these species. Significantly, a huβc S585G mutant was unable to promote haemopoietic cell survival in response to IL-3. These results identify a new mechanism by which cytokine receptors are able to couple to downstream signalling pathways that regulate cell survival. An approach was developed and optimised to analyse specific GM-CSF-mediated responses in monocytes/macrophages expressing wildtype or mutant huβc, (including huβc S585G that was defective in regulating survival). Bone marrow-derived muβc -/-;muβIL-3 -/- monocytes/macrophages were retrovirally transduced with constructs expressing wildtype or mutant huβc, along with huGMRα, then purified by FACS. Two assays were established to measure effector functions in the transduced monocyte/macrophages; (1) a flow cytometry assay for ROS production, and (2) an assay for phagocytosis. The capacity for GM-CSF to prime (i.e. enhance effector functions) ROS production and phagocytosis was investigated in huGMRα-transduced monocytes/macrophages. Our results have identified two key residues in the cytoplasmic domain of βc subunit: Tyrosine 577 (required for huβc interaction with the adaptor protein Shc) and serine 585 (required for 14-3-3 association), that are essential for the ability of GM-CSF to regulate key effector functions in monocytes/macrophages. These novel findings are significant in that they establish a molecular link between the GM-CSF/IL-3/IL-5 receptor and the regulation of both haemopoietic cell survival and inflammatory responses, and therefore have important implications in our understanding of inflammatory diseases such as rheumatoid arthritis and asthma.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 200