Exploring the role of the small GTPase Rho in T-lymphocyte biology

Rho, a member of the small GTPase superfamily, acts as a molecular switch cycling between an inactive GDP bound state and an active GTP bound form and has been reported to regulate many cellular processes. In the present thesis the role of Rho in thymocyte development has been examined. The bacterial enzyme, C3-transferase from Clostridium botulinum selectively ADP-ribosylates Rho within its effector-binding domain and thereby abolishes its biological function. Previous work has used the proximal p56lck promoter to drive thymocyte-specific expression of C3-transferase generating transgenic mice with the first thymocyte progenitors and subsequent subsets devoid of Rho function. Rho inactivation severely impaired the production of normal numbers of thymocytes and peripheral T cells in the lck-C3 transgenic mice. The main problem caused by lack of Rho function was that early thymocyte progenitors underwent massive apoptosis in the absence of Rho function. An initial aim of the present thesis was to extend analysis of Ick- C3 transgenic mice to gain greater insight into the molecular basis for the regulation of cell survival by Rho. In the lck-C3 transgenic mice the survival defect is caused by loss of Rho function in the earliest T-cell progenitors. To study Rho function in later stages of T cell development the CD2 antigen locus control region (CD2-LCR) was used to generate a series of transgenic mice in which C3 transferase is not expressed in the earliest progenitors but is targeted to a later stage of thymic development, the pre-T cell population. Elimination of Rho at this later stage results in a differentiation block of pre-T cells, but no apparent survival defect and has led to the elucidation of an additional role for Rho; as a controlling intracellular switch for the critical thymic checkpoint of TCR (3 selection. Cell survival is controlled by a complex network of signals mediated by death- domain containing receptors, Bcl-2 family members and the tumour suppressor p53. Studies of lck-C3 transgenic mice described in the present thesis have established that Rho controls survival of thymocytes by both p53 dependent and independent signalling mechanisms. The link between Rho and the tumour suppressor p53 was intriguing because during routine maintenance of the lck-C3 transgenic mice, it was observed that the mice were becoming sick and dying prematurely. Analysis revealed that loss of Rho function in the thymus was associated with the development of aggressive thymic lymphoma. These observations suggest that Rho itself may be involved in the suppression of malignancy in the thymus. The results presented in the thesis highlight the diversity of the roles of Rho within T cell biology. One way in which such diversity could arise is by this GTPase interacting with a number of downstream effectors. In an attempt to identify Rho effector molecules within T lymphocytes, a protein affinity chromatography approach was taken to purify proteins that can interact with activated GTP bound recombinant RhoA. These experiments identified the serine kinase PRK-1 and the actin regulatory protein mDia-1 as putative Rho effector molecules within T lymphocytes