Functional characterisation of the drosophila protein tyrosine phosphatase dPTP61F

The insulin signalling pathway serves an important role in many biological processes, including the maintenance of glucose homeostasis, protein synthesis, cell growth, and proliferation. The negative regulation of insulin signalling is mediated to a large extent by protein tyrosine phosphatases, including the homologous proteins T cell protein tyrosine phosphatase (TCPTP) and protein tyrosine phosphatase-1B (PTP1B). As deregulation of insulin signalling is involved in the pathogenesis of many diseases, in particular type 2 diabetes, it is important to improve understanding of the regulation of insulin signalling by PTP1B and TCPTP. To this end, the function of the Drosophila melanogaster homolog of PTP1B and TCPTP, dPTP61F, is studied. This thesis describes the characterisation of dPTP61F in the regulation of Drosophila insulin signalling. Using RNA interference against dPTP61F in S2 cells it was demonstrated that dPTP61F is a negative regulator of InR phosphorylation in vitro. Co-overexpression experiments with the Drosophila InR confirmed that dPTP61F is capable of negatively regulating insulin signalling in vivo. Furthermore, an association of dPTP61F with the adaptor protein Dock was shown to improve the efficiency of dPTP61F-mediated InR dephosphorylation. The involvement of dPTP61F in the regulation of insulin signalling may influence growth as overexpression of dPTP61F reduces both cell size and proliferation. Imprecise P-element excision was undertaken to generate a loss-of-function allele of dPTP61F. A partial mutant allele lacking two of four dPTP61F isoforms was generated and displays phenotypes including decreased median lifespan and reduced female fecundity, identifying novel roles of dPTP61F. Increased apoptosis and a reduction in the number of vitellogenic egg chambers is a likely cause of the reduced fecundity. This thesis investigated the involvement of several biological pathways, including insulin signalling, in mediating the decrease in fecundity. This thesis also examined whether human PTP1B and TCPTP can function in Drosophila. Misexpression studies confirmed that PTP1B and TCPTP can negatively regulate Drosophila insulin signalling. In addition, the nuclear localised isoform of TCPTP, TC45, can rescue the fecundity defect of the dPTP61F partial loss-of-function mutant. These studies describe the role of dPTP61F in the regulation of insulin signalling and additional biological processes and give insight into the importance of subcellular localisation in mediating substrate specificity and selectivity of PTPs