Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.600751
Title: The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases
Author: Pavic, Karolina
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2013
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Abstract:
Abnormalities in the coordinated activities of protein phosphatases (PPs) and protein kinases (PKs) contribute to the development of many diseases. Phosphatase of regenerating liver (PRL)-3 and Vaccinia H1-Related (VHR) are two members of the protein tyrosine phosphatase (PTP) family shown to be involved in cancer. PRL-3 is a member of the PRL phosphatases containing a unique post-translationally modifiable prenylation CAAX motif at the carboxy (C)- terminal end. There is an immense body of evidence to support a role for PRL-3 in the development of various types of cancer and in progression to metastasic disease. However, many questions are still pending, especially with respect to the identity of physiological substrates, and interacting partners in general, of PRL-3. VHR is a model for a group of atypical dual specificity protein phosphatases (DUSPs) with a role in cell cycle progression. Only a few of VHR’s physiological substrates have been reported to date and there are very few studies addressing its regulation and physiological role(s). Generally, in order to isolate and identify transient phosphatase-substrate interactions, substrate-trapping mutants of PTPs are employed. Mutants which can function as substrate traps have the ability to recognise and bind substrates, yet they lack functionality of the key catalytic residues and cannot efficiently process the hydrolysis of the substrate. However, it is acknowledged that the efficiency of such standard substrate-trapping mutants of PTPs is low. In this work, the expanded genetic code approach was applied to develop more efficient substrate trapping variants of the PTPs by incorporating the photocross-linkable amino acid para-benzoylphenylalanine (pBPA). The concept was optimised for PRL-3 and VHR, and for both proteins, pBPA-containing variantswere expressed at excellent yields and were highly purified. By utilizing the photo-cross-linkable F68pBPA variant of VHR, dimerisation of VHR was detected in an in vitro ultraviolet (UV) exposure-mediated crosslinking assay. VHR dimerisation was further demonstrated to be a potential novel regulatory mechanism for VHR, having a negative effect on the catalytic activity of the protein. A specific region in VHR known as the variable insert segment was pinpointed as a region in the protein, which is either at the dimer interface or heavily contributing to dimeric association. Furthermore, the intrinsic ability of VHR to self-associate was also demonstrated by complementary methods. For PRL-3 it was demonstrated that its D72pBPA variant could recognise and bind to lipids, with a stronger signal detected in the UV-exposed sample, and without altering the lipid binding profile with respect to the native protein. Lastly, the potential of exploiting photo-cross-linkable variants of the PTPs was also demonstrated by incubating PRL-3 variants, with or without selectively introduced pBPA, with mammalian cell lysates, followed by UV exposure. Western blot analysis detected new bands corresponding to covalently crosslinked PRL-3-protein complexes. Future work in our laboratory will follow up on these newly identified interactions.
Supervisor: Kohn, Maja; Krijgsveld, Jeroen; Lemke, Edward Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.600751  DOI: Not available
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