Molecular Analysis of Signalling Pathways: Understanding their Roles in Development and Disease
Our laboratory is studying the molecular pathways via which signals are transmitted from receptors to the cytoplasm and ultimately the nucleus of cells where gene transcription is regulated. We are applying a multi-disciplinary and multi-organismal approach in elucidating the regulatory components involved in signalling via protein phosphorylation with the aim of identifying novel therapeutic targets for treatment of diseases such as cancer, diabetes and neurological disorders.
Certain signals can stimulate cells to trigger apoptotic pathways, while others induce protective effects. Clearly, the propensity of a cell to live or die is an important factor in tumorigenesis and many cancers are caused by a reduction in the tendency of a cell to commit suicide. Desensitization of pro- apoptotic circuitry or up-regulation of survival signals will not only distort the longevity of cells but will also modulate the efficacy of treatments. We have been studying the role of the phosphatidylinositol 3' kinase pathway. This signalling system generates 3' phosphorylated phospholipids at the cell membrane that act to recruit a series of protein kinases leading to the activation and translocation of an enzyme termed protein kinase B (PKB/Akt). PKB then phosphorylates key proteins within the cell, many of which are involved in regulating cell viability. One of these targets is GSK-3, the same enzyme that is regulated by the Wnt pathway (see below). Using transgenic mice and mutant flies we have investigated the physiological significance of ativation of PKB in T cells and the mammary gland. In collaboration with Dr. Armen Manoukian , we identified a new target of the Drosophila enzyme termed Trachealess which is a master switch for development of the breathing system of fruit flies . Using inducible activation mutants, we have probed the mechanisms by which PKB and its upstream kinase, PDK1, are regulated and the consequences of their chronic stimulation. We are employing similar approaches to investigate the functions of a related protein kinase termed Serum and Glucocorticoid-dependent Kinase 3 (SGK3) by generating mammary gland transgenic mice and by understanding its mechanism of activation. These animals develop mammary tumours with high penetrance, unlike the activated PKB mice. We are determining the molecular targets of SGK3 that result in its oncogenic properties.
Wnt/wingless signalling and cell fate determination:
The Wnt pathway has been highly conserved during evolution and mediates cell fate decisions and differentiation. The remarkable degree of similarity in the structure and function of of components of this pathway has allowed use of genetically tractable organisms such as yeast, mould and fruit flies to determine its function in complex processes. The pathway involves several cancer related genes including the tumour suppressor, APC, and beta-catenin. A key regulatory component of this system is a protein kinase termed Glycogen Synthase Kinase-3 (GSK-3). The fruit fly orthologue is termed Shaggy or Zeste-White3 and this gene is one of the primary interests of Dr. Manoukian . We have generated knocout mice that lack each of the two genes that encode GSK-3 in mice (e.g GSK-3beta; and GSK-3alpha and have also generated mice that harbour conditional alleles of these genes. Using embryonic stem cells that are defective for both copies of GSK-3alpha and beta, we have teased apart the molecular mechanisms of specificity of this enzyme to understand how it contributes to multiple signalling pathways without "crossing wires". Embryonic stem cells that lack all four alleles of GSK-3 show a profound block in differentiation capacity. We are exploiting this property to expand progenitor cells from mice in which the two GSK-3 genes have been engineered as conditional alleles towards the goal of developing methodologies for culturing specific cell types for repair of lesions and regenerative medicine. This approach is also allowing assessment of the impact of suppressing differentiation pathways on the potential and frequency of tumorigenesis as well as the impact of various signalling pathways (including the Wnt, Notch and PI3K systems) on stem cell pluripotency and differentiation.
Stress-activated protein kinase pathways:
This pathway is induced by a variety of cellular stresses and impinges on several transcription factors. We are determining the physiological roles for this transductory system using small molecule inhibitors, inducible activation systems and genetically modified cells to understand the functions for different components. In particular, we are interested in the high conservation of splice variants of these protein kinases as well as their selective physiological functions.Together, these studies provide insight into the functions and detection of the control circuits which must be thwarted to escape normal growth regulation and thus are providing novel targets for therapeutic intervention in a variety of human diseases.