Stach, L; (2013) Functional analyses of pSer and pThr binding domains. Doctoral thesis , UCL (University College London).

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Abstract

Since the discovery of the phosphotyrosine binding SH2 domain, many classes of phospho-recognition domains have been described which mediate many of the diverse cellular functions of protein kinases. Among those, FHA domains are unique in their ability to exclusively recognise pThr epitopes. The genome of the human pathogen Mycobacterium tuberculosis encodes 5 FHA domains, along with 11 Ser/Thr protein kinases. In the first part of this thesis, it is shown that Ser/Thr protein kinase PknB phosphorylates a threonine residue in an intrinsically unstructured region of protein FhaA. FhaA contains an FHA domain through which it interacts with and presumably inhibits MviN, a muropeptide flippase essential for cell-wall synthesis. Upon phosphorylation, the FHA domain binds the pThr epitope in an intra-molecular interaction occluding the MviN binding surface and alleviating its inhibition. Although the pThr-FHA interaction is relatively weak and nonspecific, the phosphorylated molecule nonetheless assumes a ‘closed’ conformation 99% of the time and is therefore able to outcompete the 2 orders of magnitude stronger bimolecular FHA-MviN interaction. In the second part, the phospho-binding capabilities of the human PIH1D1 protein were characterised. PIH1D1 has been shown to interact with a central chaperone assembly comprising the R2TP complex and Hsp90. It has also been shown to interact with co-factor Tel2 in a phospho-dependent manner essential for the stability of the ‘giant’ PI3-kinase-like kinases mTOR and SMG1. PIH1D1 is shown to function as a novel phospho-reader domain with a consensus binding sequence of D-pS-D-D, agreeing well with the substrate specificity of casein kinase 2. A mutant that abolishes phospho-binding was identified and used in binding experiments which showed that PIH1D1 interacts with the chaperone complex phospho-independently and that its phospho-binding capacity is utilised to recruit a subset of CK2 substrates to the chaperone complex.

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