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

Abnormalities in the coordinated activities of protein phosphatases (PPs) and<br/>protein kinases (PKs) contribute to the development of many diseases.<br/>Phosphatase of regenerating liver (PRL)-3 and Vaccinia H1-Related (VHR) are<br/>two members of the protein tyrosine phosphatase (PTP) family shown to be<br/>involved in cancer. PRL-3 is a member of the PRL phosphatases containing a<br/>unique post-translationally modifiable prenylation CAAX motif at the carboxy<br/>(C)- terminal end. There is an immense body of evidence to support a role for<br/>PRL-3 in the development of various types of cancer and in progression to<br/>metastasic disease. However, many questions are still pending, especially with<br/>respect to the identity of physiological substrates, and interacting partners in<br/>general, of PRL-3. VHR is a model for a group of atypical dual specificity<br/>protein phosphatases (DUSPs) with a role in cell cycle progression. Only a few<br/>of VHR’s physiological substrates have been reported to date and there are<br/>very few studies addressing its regulation and physiological role(s).<br/>Generally, in order to isolate and identify transient phosphatase-substrate<br/>interactions, substrate-trapping mutants of PTPs are employed. Mutants which<br/>can function as substrate traps have the ability to recognise and bind<br/>substrates, yet they lack functionality of the key catalytic residues and cannot<br/>efficiently process the hydrolysis of the substrate. However, it is acknowledged<br/>that the efficiency of such standard substrate-trapping mutants of PTPs is low.<br/>In this work, the expanded genetic code approach was applied to develop more<br/>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.<br/>By utilizing the photo-cross-linkable F68pBPA variant of VHR, dimerisation of<br/>VHR was detected in an in vitro ultraviolet (UV) exposure-mediated crosslinking<br/>assay. VHR dimerisation was further demonstrated to be a potential<br/>novel regulatory mechanism for VHR, having a negative effect on the catalytic<br/>activity of the protein. A specific region in VHR known as the variable insert<br/>segment was pinpointed as a region in the protein, which is either at the dimer<br/>interface or heavily contributing to dimeric association. Furthermore, the<br/>intrinsic ability of VHR to self-associate was also demonstrated by<br/>complementary methods.<br/>For PRL-3 it was demonstrated that its D72pBPA variant could recognise and<br/>bind to lipids, with a stronger signal detected in the UV-exposed sample, and<br/>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<br/>introduced pBPA, with mammalian cell lysates, followed by UV exposure.<br/>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.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2013

Author dc:creator

• Pavic, Karolina

Advisors dc:contributor.advisor

• Keyse, Stephen
• Kohn, Maja
• Krijgsveld , Jeroen
• Lemke, Edward

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/39cd7ecc-d09b-4295-933e-725538437046

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/39cd7ecc-d09b-4295-933e-725538437046