ASPP1 and ASPP2 link the Ras and p53 signalling pathways

Abstract

In this thesis, the regulation of ASPP1 and ASPP2 was investigated. ASPP1 and ASPP2 are p53 co-activators that can specifically induce p53 dependent apoptosis but have no effect on p53-dependent cell cycle arrest. Both ASPP1 and ASPP2 contain a Ras-association domain in their amino terminal regions. ASPP1 can bind activated Ras directly via its amino terminal region in vitro, and both endogenous ASPP proteins bind endogenous Ras in vivo after stimulation of cells with serum and growth factors. Oncogenic H-RasV 12 and K-RasV 12 stimulate ASPP1 and ASPP2 pro-apoptotic activity in a p53-dependent manner and can also stimulate ASPP2 co-activation of the p53 family members, p63 and p73. These results suggest that ASPP1 and ASPP2 are novel Ras effector proteins. Ras is upstream of several effector pathways. One of its downstream effector pathways, Raf-MEK-MAPK, can activate ASPP1 and ASPP2. MAPK phosphorylates ASPP2 in vitro, and both ASPP1 and ASPP2 in vivo at serines 746 and 827, respectively. ASPP1 and ASPP2 phosphorylation by MAPK results in an increase in their ability to co-activate p53. Additionally, MAPK phosphorylation of ASPP2 leads to increased ASPP2 protein levels, suggesting that MAPK can regulate ASPP2 by modulating its protein stability. ASPP1 and ASPP2 deletion fragments were used to examine the regulation of ASPP proteins. Amino-terminus fragments were shown to increase full-length ASPP activity when co-transfected. Moreover, PKA was also found to be a regulator of ASPP2 and was shown to phosphorylate ASPP2 in vitro. Forskolin, a stimulator of PKA, could enhance ASPP2 activity. The results provide the first insight into these novel mechanisms by which ASPP activity may be regulated