Burdon, Tom

Maciver, Sutherland

Ho, Daniela Gattegno

2010-11-10T16:42:55Z

2010-11-10T16:42:55Z

2009

Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.

http://hdl.handle.net/1842/4252

en

The University of Edinburgh

Gab1β

embryonic stem cells

stem cells

AKT

ERK

Characterising the function of a novel embryonic stem cell-associated signal transducer, Gab1β

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy