Copy of author's previously published work inserted. Includes bibliographical references (leaves 160-182). Concentrates mainly on the characterisation of the molecular mechanism of action of the tec protein tyrosine kinase using biochemical and genetic approaches.

Bibliography: leaves 139-165.

Bibliography: leaves 139-165. / ix, 183 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2001

Copy of author's previously published work inserted. / Includes bibliographical references (leaves 160-182). / xi, 182 leaves, [57] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Concentrates mainly on the characterisation of the molecular mechanism of action of the tec protein tyrosine kinase using biochemical and genetic approaches. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?

"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. / Includes bibliographical references (leaves 170-214). / xiv, 221 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-170).

Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signaling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative variant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that was first reported as a fusion partner of nucleophosmin in Anaplastic large cell lymphoma in 1994. ALK is involved in myriad of cancers including neuroblastoma which is the most common extracranial solid tumor affecting young children. It arises in the neural crest cells of sympathetic nervous system origin and is responsible for 12% of all childhood cancer deaths. Several point mutations in ALK have been described in both familial and sporadic neuroblastoma. With the aim to understand the role of ALK in neuroblastoma further, we investigated the point mutations in ALK reported in patients. Using cell culture based methods and Drosophila as a model organism; we first characterized these mutations under three broad categories: 1) Ligand independent mutations that were constitutively active, 2) Kinase dead mutation and 3) Ligand dependent mutations that behaved as inducible wild type. Further, to understand the activation mechanism of ALK, we constructed mutations that could potentially alter ALK’s conformation based on the available crystal structure. From the data generated, we were able to provide a new perspective to the activation of full length ALK receptor. This was more in line with activation mechanism of insulin receptor and different from that suggested for ALK fusion protein. From a clinical point of view, all the mutations in the study were blocked to different degrees using the ALK inhibitor, crizotinib. Lastly, we identified potential downstream targets of ALK using phosphoproteomics. From the various targets identified, we focused on STAT3 and confirmed its role as a mediator in ALK initiated MYCN transcription. We showed that STAT3 inhibition led to reduction of MYCN levels and thereby identifying it as a potential therapeutic target in neuroblastoma. Overall, our study highlights clinical relevance of ALK mutations in neuroblastoma and from a basic biology viewpoint; it reveals important mechanistic insight into receptor activation.

Mutation and over-expression of Receptor Tyrosine Kinases (RTKs) or the proteins they regulate serve as oncogenic drivers in diverse cancers. RTKs catalyze the transfer of phosphate from ATP to the hydroxyl group on tyrosine. The proximal stretch of amino acids including this post translational modification is then able to be recognized by SH2 and PTB domains. Chapter 1 details our work to better understand RTK signaling and its link to oncogenesis using protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 out of the 53 Receptor Tyrosine Kinases. Chapter 2 expands upon this work to study the next layer of binding among SH2 and PTB domain-containing adaptor proteins themselves. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins driving oncogenesis, including both receptors and adaptor proteins, tend to be highly interconnected via a network of SH2 and PTB domain-mediated interactions. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in these well-studied signaling networks. Despite the extensive work presented here on experimentally determining interactions, we nevertheless are unable to keep up with the discovery of new sites of tyrosine phosphorylation by high throughput mass spectrometry as well as their mutation in cancer discovered by next generation tumor sequencing approaches. Chapter 3 introduces work in progress to build a unified predictive model of SH2 domain interactions via integration of diverse data sets of binding as well as crystal structures of domain-peptide interactions. This model will enable researchers discovering new phosphorylation events or mutations to be able to predict potential interaction partners and thereby elucidate novel functional mechanisms. / Chemistry and Chemical Biology