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Author Liau, Nicholas

Title Structural and biochemical characterisation of the regulation of Janus Kinase signalling

Published 2017

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 UniM INTERNET Thesis    AVAILABLE
Physical description 1 online resource
Thesis notes Thesis (PhD thesis)-- Medical Biology 2017
Summary The cytokines are a group of extracellular signalling proteins which stimulate a large variety of cellular processes, including the proliferation and differentiation of blood cells, and the up-regulation of the immune system in response to infections. Cytokines are able to bring about such changes by binding to the extracellular domains of specific cytokine receptors displayed on the target cell surface. Cytokine stimulation causes a conformational change to the receptor, activating the Janus Kinases (JAKs), which are attached to the intracellular portion of the receptor. The JAKs are tyrosine kinases, and are able to propagate a signal by phosphorylating downstream substrate proteins, including the Signal Transducers and Activators of Transcription (STATs). Once phosphorylated, the STATs enter the nucleus where they can cause the upregulation of particular gene sets. Whilst cytokine signalling is important for normal cellular function, overactive signalling can be detrimental. Mutations to the JAKs causing overactive signalling have been associated with a group of diseases known as the myeloproliferative neoplasms (MPNs), as well as some cancers. The cell has developed mechanisms to ensure JAK signalling is only active when required. The JAKs are switched off under resting conditions, and only become activated in response to cytokine signalling except for some constitutively active JAK disease-causing mutants. Other proteins are also able to downregulate the JAKs, including the Suppressors of Cytokine Signalling (SOCS) proteins, which are themselves upregulated in response to cytokine signalling to ensure that JAK activation is only transient. In this thesis, SOCS1 was shown to potently downregulate JAK catalytic activity. Structural experiments showed that the short Kinase Inhibitory Region (KIR) of SOCS1 is able to directly inhibit JAK kinase activity by blocking access of substrates to the JAK catalytic site. SOCS1 also contains an SH2 domain, and biochemical experiments showed this domain targets a number of specific phosphotyrosine-containing motifs on the intracellular portions of certain cytokine receptors and on JAKs themselves. Few structural or biochemical studies of full-length JAK (flJAK) proteins have been performed owing to the difficulty of obtaining purified, active, recombinant protein. Following an extensive screen of expression, purification and construct optimisation, methods were successfully developed to obtain high yields of pure, active flJAK1 protein. A biochemical characterisation of flJAK1 was undertaken, demonstrating that the full-length protein binds ATP more tightly, and is catalytically less active than the kinase domain alone showing that other domains regulate kinase activity. Finally, structural studies of full-length JAK1 were undertaken. A complex of flJAK1, SOCS3 and a fragment of the gp130 receptor was successfully crystallised, and crystals diffracted to approximately 8Å resolution, though the three-dimensional structure could not be solved. Low-resolution structural studies showed that the complex adopts an overall elongated conformation in solution. The flJAK1-SOCS3-gp130 complex may be amenable to further high-resolution structural studies. Together, these studies have provided novel insights into how the JAKs are regulated by exogenous proteins such as SOCS1, as well as how they may be activated in response to cytokine stimulation.
Subject Janus Kinase JAK Suppressor of Cytokine Signalling 1 SOCS1 structural biology