The Development of Photo-crosslinkable Trapping Mutants as Tools to Investigate the Interactions of Protein Tyrosine Phosphatases

  • Karolina Pavic

    Student thesis: Doctoral ThesisDoctor of Philosophy


    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.
    Date of Award2013
    Original languageEnglish
    Awarding Institution
    • European Molecular Biology Laboratory
    SupervisorStephen Keyse (Supervisor), Maja Kohn (Supervisor), Jeroen Krijgsveld (Supervisor) & Edward Lemke (Supervisor)

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