Rodriguez-Viciana, Pablo; (1998) Role of phosphatidylinositol 3-kinase as an effector of Ras. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ras genes are found mutated in about 30% of all human malignancies, being one of the most frequent mutations in human cancer. They code for 21 kiloDalton GTPases that act as molecular switches at a critical crossroad in the control of proliferation, differentiation and many other cellular processes. When in its active, GTP-bound state, Ras can interact with its downstream targets or effectors to carry out its biological function. The best characterised Ras effector is the Raf serine/threonine kinase, through which Ras activates the MAP kinase cascade. The work presented in this thesis, describes the identification of Phosphoinositide 3-kinase (PI 3-kinase) as an additional Ras effector. Ras, when bound to GTP, interacts through its effector domain with the p1 10 catalytic subunit of PI 3-kinase. This interaction results in stimulation of the lipid kinase activity of PI 3-kinase, both in intact cells and in an in vitro reconstitution system. Furthermore, expression of the dominant negative mutant N17 Ras, inhibits EGF and NGF-induced activation of PI 3-kinase in intact PC 12 cells. In order to study the contribution of PI 3-kinase to Ras function, mutations in the effector domain of Ras have been generated, that selectively impair the ability of Ras to interact with different effectors. The Y40C mutation allows Ras to interact only with PI 3-kinase, T35S and D38E mutants interact only with Raf and the E37G mutant interacts only with RalGDS. These partial loss of function mutants, together with activated and dominant-negative versions of PI 3-kinase and other effectors, have been used to study the effects of the different Ras effector pathways in several cell systems. It is shown that Ras needs to activate simultaneously several pathways in order to efficiently transform fibroblasts. Although no single pathway is sufficient, PI 3- kinase makes a critical contribution to Ras transforming potential: inhibition of PI 3- kinase function inhibits loss of contact inhibition, loss of anchorage-dependence for growth and morphological transformation induced by Ras. PI 3-kinase, through the activation of the Rac GTPase, is also a critical mediator of the effects of Ras on the actin cytoskeleton. In addition, evidence is presented that activation of PI 3-kinase may play a role in protecting tumour cells harbouring Ras mutations from programmed cell death. The results suggest that the PI 3-kinase pathway may be an attractive novel target for therapeutic intervention in the treatment of tumours where Ras is involved.

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