| Popis: |
Ras proteins are GTPases that are molecular central hubs for propagation of intracellular signals that are involved in diverse processes, including cell differentiation and proliferation. The four main Ras isoforms, HRas, NRas and the two KRas splice variants, KRasA and KRasB, are highly homologous and conserved proto-oncogenes that constitute a paradigm of cellular transformation. While all RAS genes are commonly mutated in human cancers, different isoforms couple to distinct tumours and the mutations in KRAS constitute the majority (~86%) of all RAS mutations. Intriguingly, KRasB is the only Ras isoform essential for normal embryonic growth in the mouse. Such discrepancy of Ras isoform contribution to different cancers and the uniquely essential role of KRasB in normal development may stem from the distinct spatiotemporal expression of Ras isoforms and their differential coupling to downstream effectors. However, to date there has been no comprehensive quantitative comparison of Ras isoform expression across various tissues throughout development. Moreover, there are no studies that compared Ras isoform-specific signalling in an endogenous context. Therefore, this thesis aims to provide the first complete map of Ras isoform expression during development and the first comparison of endogenous Ras isoformspecific signalling. In the first part of this work, quantitative real-time RT-PCR was used to measure Ras isoform transcript levels in mouse embryonic stem cells (ESCs) and in a panel of embryonic, postnatal and adult mouse tissues. KRasB was found to be the most highly expressed isoform, whereas KRasA was shown to be the most dynamically regulated. Transcript copy number does not necessarily correlate with protein copy number; therefore, protein standard absolute quantitation mass spectrometry was used to accurately measure tissue Ras protein levels. In contrast to the qRT-PCR data where KRasB was 5-10-fold higher expressed than any of the other isoforms, protein abundance levels were found to be similar for the Ras isoforms. The mechanistic basis for this and the implications for models of isoform-specific Ras association with specific cancers are discussed. In the second part of this thesis, isogenic SW48 human colorectal cancer cell lines identical except for the presence of an activating G12V mutation in each of the three RAS gene loci were utilised to study endogenous Ras signalling. The results revealed isoform-specific coupling to canonical Ras effector pathways for example, HRas was the most potent activator of downstream MAPK and PI3K pathways. These data represent the basis for planned network biology studies to model Ras isoform-specific signalling. Together, this study provides the first most complete approaches for studying baseline Ras isoform expression and signalling in an endogenous context. The results guide our understanding of Ras isoform-specific network biology, coupling to distinct human cancers and involvement in normal development. |
