The ability of a cell to respond to extrinsic stimuli critically depends on its ability to regulate specific intracellular protein - protein interactions in a reversible manner and allow the temporal - spatial characteristics of the signal to be accurately transduced to downstream targets. Growth factor and cytokine receptors provide a link by which extracellular stimuli are propagated within the cell to accomplish specific cellular functions. Ligand - stimulation of these receptors activates cascades of intracellular events that transduce the signals that lead to a variety of cellular responses. The specificity of these signals and the fidelity with which they are communicated within the cell are critical for the fate of an organism as deregulation or misbalance of signalling networks is commonly associated with a wide range of pathologies and diseases. Cytokines are important regulatory proteins that regulate diverse cellular functions through their ability to bind to specific cell surface receptors. Most cytokines are pleiotropic effectors that regulate multiple cellular functions. For example, many cytokines can regulate diverse biological activities such as cell survival, proliferation and differentiation and in many cases these different biological activities can be independently regulated. The regulation of pleiotropic biological responses is mediated through the modulation of multiple intracellular signalling pathways. These pathways often present a high level of redundancy in terms of the biological functions that they control. However, pleiotropic cytokines have the ability to independently activate signalling pathways that lead to the regulation of specific biological functions such as survival, proliferation, differentiation or activation. The molecular mechanisms by which cytokines can regulate pleiotropic biological responses from the activation of a limited number, often redundant, of intracellular signalling pathways have not been fully resolved and remains one of the most important unanswered questions in cell biology. In particular, proteins and molecular mechanisms responsible for specifying different biological responses remain largely unidentified. In many cases, activation of multiple signalling pathways and integration of the signals they transduce is needed in order to modulate a biological function. One important mechanism by which signalling pathways are assembled within the cell is through the action of protein scaffolds that contain phosphotyrosine ( e.g. SH2, PTB ) or phosphoserine / threonine ( e.g. 14 - 3 - 3, WW, FHA, PBD, BCRT ) binding modules. Interestingly, although phosphotyrosine and phosphoserine / threonine - dependent signalling pathways are highly integrated within the cell, scaffold proteins containing both phosphotyrosine and phosphoserine or phosphothreonine - binding domains ( i.e. SH2 / PTB and WW / FHA / PBD / BCRT ) have not been identified. The broad aim of this thesis is to study the fundamental molecular mechanisms by which cytokines, through the binding of cell surface receptors, are able to activate and integrate signalling pathways that regulate and specify cellular responses. In particular, these studies examine the role of the 14 - 3 - 3 family of adaptor proteins in the assembly of signalling networks that couple the activated receptors of the haematopoietic cytokines IL - 3, IL - 5, and GM - CSF to downstream signalling targets and specific cellular functions such as survival and proliferation. The specific aims of this thesis are to examine the composition, molecular mechanisms of assembly and functional roles of signalling complexes that use the adaptor or scaffold protein 14 - 3 - 3 and are important for signal transduction in response to GM - CSF. This work shows that the phosphoserine / threonine - binding scaffold protein 14 - 3 - 3 ζ, previously reported to bind to Ser585 of the GM - CSF receptor, undergoes tyrosine phosphorylation. Using a panel of 14 - 3 - 3 ζ mutants a particular tyrosine residue, Tyr179, was found to be critical for the binding of the SH2 domain of Shc, the assembly of a PI3K signalling complex, the activation of the Akt / PKB signalling pathway and the control of cell survival in response to GM - CSF stimulation. Tyr179 of 14 - 3 - 3 ζ was also found to be important for specifying GM - CSF - mediated biological responses as it was found to play an important role in the control of cell survival versus cell proliferation. Furthermore, it was found that 14 - 3 - 3 ζ is able to simultaneously bind to Ser585 of the GM - CSF receptor and recruit Shc and PI3K through Tyr179, thus integrating phosphoserine / threonine and phosphotyrosine / dependent signalling pathways. The findings described in this thesis helped to identify a novel mechanism by which cytokine receptors achieve both integration in signalling and specificity in biological outcomes. The discovery that phosphoserine / threonine - binding proteins ( i.e. 14 - 3 - 3 ) are themselves tyrosine phosphorylated and able to recruit phosphotyrosine - binding molecules provides a new insight into how intracellular signal integration is achieved. Understanding how signal transduction is carried out within the cell is paramount to successful drug development in many therapeutic areas. The new insights in GM - CSF signalling provided by this work may help to successfully develop treatments to target diseases such as asthma, rheumatoid arthritis and leukaemia, where GM - CSF appears to play a pathogenic role. / Thesis (Ph.D.)--School of Medicine, 2006.
| Identifer | oai:union.ndltd.org:ADTP/263692 |
| Date | January 2006 |
| Creators | Felquer, Fernando Augusto |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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