Sahai, Erik Anand; (1998) Functional analysis of RhoA and its effector molecules. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes studies on the small G protein RhoA and how it regulates a diverse range of cellular processes. The introduction outlines some of the general principles and mechanisms of how cells respond to extracellular stimuli, in particular the role of small G proteins as molecular switches. The last part of the introduction deals specifically with the small G protein RhoA, the aspects of cell behaviour that it controls and its potential molecular targets. Chapters 2 and 3 describe the generation of RhoA mutants that are unable to interact with specific target molecules. The ability of these mutants to bind target molecules has been correlated with their ability to regulate the cytoskeleton, gene expression via the transcription factor SRF, and cell transformation. These analyses revealed that regulation of SRF can occur in the absence of cytoskeletal changes and transformation by RhoA correlates with binding of the kinase ROCK, not SRF activation. The role of the kinases, PKN and ROCK, as RhoA target molecules is also investigated by overexpressing mutant versions of the proteins. Chapter 4 describes the use of a pharmacological inhibitor of ROCK, Y-27632, to investigate its role as RhoA target, in particular, its role in transformation. These studies confirm the findings of Chapter 3: ROCK is required for RhoA mediated transformation and cytoskeletal changes but not signalling to SRF. Chapters 5 and 6 describe the isolation of a novel RhoA binding clone using the yeast two-hybrid system and its characterisation. A RhoA mutant specifically unable to interact with the novel clone is described and tested functionally. Chapter 7 discusses the work in this thesis. Models are proposed for the regulation of the cytoskeleton, transcription and transformation by RhoA. Possible criticisms of the approaches used are evaluated. The role of ROCK in regulation of the cytoskeleton and transformation and possible links between the two are discussed.

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