Medical Biology - Theses
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ItemThe interaction between Janus Kinase and cytokine receptors( 2023-07)Janus Kinase (JAK) is a multidomain, non-receptor tyrosine kinase that is essential for signal transduction in the JAK/STAT signalling pathway and regulates biological activities such as proliferation, differentiation, and apoptosis. The JAK/STAT pathway is particularly important in driving haematopoiesis and the immune response. JAK must be constitutively associated with the intracellular domain of a cytokine receptor to carry out its function and it binds to two main sites termed box 1 and 2 on a receptor using its FERM-SH2 domains, which form a single structural motif. There are more than 50 cytokines and receptors in humans, and they are largely conserved through vertebrate evolution. Each cytokine receptor consists of at least two protein chains and whilst some receptors are homodimeric, most are heterodimers and higher-order oligomers. It is thought that each chain can interact with only a single JAK however there are exceptions. To date, the interaction between only five receptors bound to its associated JAK have had their structure solved. The importance of the JAK/Receptor interaction is highlighted by the fact that it is this interaction which goes wrong in a number of diseases such as SCID (severe combined immunodeficiency), due to loss of this interaction and cancers where this interaction leads to aberrant activation of the JAK/STAT pathway. This research study aims at investigating the JAK/Receptor interaction at a biochemical and structural level to understand whether there are rules determining the engagement of one or more particular JAKs with a particular receptor. In chapter three, the interaction between two JAK family members and ten receptors was quantified using an SPR competition assay. Surprisingly, it was found that JAK1 and JAK2 bind weakly to all receptors studied except IFNLR1. JAKs bind to two distinct motifs on the receptor termed box1 and box2. In general, the addition of box2 increased the affinity of JAKs for receptors but even the box 1 and 2 motif combined (for all receptors apart from IFNLR1) led to a lower affinity than was expected. The minimal binding motif in IFNLR1 was identified and found to consist of the conserved motif (PxxLxF) previously identified in addition to seven amino acids upstream. The finding that most receptors bound weakly led to a hypothesis that there was a missing component in the in vitro binding assays that were conducted here. Several possibilities were considered, and these occupied the remainder of chapter three and all of chapter four. The first possibility tested was that dimerization of receptors might stabilise JAK binding to receptors. However, the results revealed it did not improve a receptor (IFNAR2) interaction with JAK1. Next, it was hypothesized that the membrane might stabilise the association between JAK and receptor as it has previously been reported to do so; this was investigated in chapter four. Using a lipid overlay assay, it was revealed that JAK1 (but not JAK2) binds to a particular phosphatidylinositol monophosphate (PtdIns(3)P). This phosphatidylinositol is enriched in early endosomes, an organelle known to be important in signalling by some cytokines. The long and charged 3-4 loop in JAK1 F3 subdomain was responsible for its interaction with this phospholipid. JAK2 had a shorter 3-4 loop and could not bind to PtdIns(3)P. However, this interaction could not be verified with a different technique. In chapter five, the structure of twelve JAK-Receptor complexes was predicted using AlphaFold. These models revealed that receptor box 1 and 2 bound to the FERM and SH2 domain of JAK respectively, as expected. The interaction between the two proteins were characterised by a largely hydrophobic motif in box 1 and likewise in box 2 with the addition of a negatively charged residue that bound to the SH2 domain in the pocket used by canonical SH2 domains to bind phosphotyrosine. Identifying and indeed predicting box 1 and 2 motifs has previously been difficult due to a lack of structural information and also due to large sequence variation in different receptors. However here we identify a canonical box 1 motif as: VPxPxxSxIxxWxP (x represents any other amino acid) and a more encompassing definition as φPxPxxSxφxxφxφ (φ represents a hydrophobic residue). In the latter half of this chapter, a powerful methodology called deep mutational scanning (DMS) was employed to determine individual residues required (in the receptor) to allow signalling. The methodology mutated every residue in the TpoR intracellular domain to every one of the 20 amino acids one-by-one. It proved to be a powerful technique to ascertain important regions in the receptor required for signalling. Not only did mutation of residues in TpoR box 1/2 inhibit signalling and support what was observed in the AlphaFold model. It also highlighted regions of the receptor with unknown (or only partially characterized) function as being crucial for signalling. In particular, it identified two regions with a likely role in interacting with the cell membrane and showed that mutation of one of these regions could lead to both gain- and loss-of-function. In addition loss of function mutations on TpoR box 1/2 that may lead to the development of diseases were identified. In summary, this study provides information on the JAK binding motifs on receptor box 1/2, their affinity and the regions of the receptor required to propagate signalling.