Molecular Mechanisms Underlying the Regulation of Protein Phosphatase-1c by the Apoptotic Stimulating Proteins of p53

Abstract

Protein phosphatase-1c (PP-1c) is a ubiquitous serine/threonine protein phosphatase regulated in part by association with a large number of different regulatory subunits. Apoptotic Stimulating Proteins of p53 (ASPPs) are a family of proteins that regulate the apoptotic function of the tumour suppressor p53. In humans, there are three family members, ASPP1, ASPP2, and an inhibitory form, iASPP uniquely overexpressed in many cancers. Whilst iASPP lacks a tetrapeptide PP-1c RVXF binding motif, this work demonstrates that iASPP interacts with PP-1c via a previously uncharacterized“RARL” binding motif. Molecular modeling and site-directed mutagenesis of PP-1c•ASPP protein complexes identified two novel modes of interaction between PP-1c and ASPP proteins. Firstly, a positively charged region of PP-1c, comprising residues Lys-260 and Arg- 261, was found to be important for binding all three ASPP family members. Secondly, the PP-1c C-terminus was shown for the first time to contain a classic type-2 SH3 poly-proline binding motif (PxxPxR), which binds directly to the SH3 domain of ASPP proteins. Surface plasmon resonance (SPR) experiments further demon- strated that a peptide derived from the PP-1c C-terminus alone binds with high anity to iASPP (K=26 nM) and ASPP2 (123 nM). The ability of the ASPP proteins to regulate PP-1c mediated dephosphorylation of p53 was examined. Gel filtration chromatography showed that binding between PP-1c, p53, and ASPP1/2 is mutually exclusive. In marked contrast, iASPP formed a stable PP-1c•iASPP•p53 trimeric complex. Dephosphorylation assays further demonstrated that formation of this complex facilitated site-directed dephosphorylation of p53 on residue Ser-15, but not Ser-37. The results from this thesis provide support for the PP-1c•iASPP complex as a valuable drug target for cancer therapies. To investigate this, over 150 marine extract leads were tested for their ability to disrupt binding of PP-1c to iASPP, from which three marine compounds were isolated and identified: sokotrasterol sulphate and two novel hippospongin-related compounds. In conclusion, this PhD thesis provides for the first time a molecular explanation for how p53 may be regulated in part through interaction with iASPP and PP-1c. This study may aid in development of novel cancer therapies that specifically target the association between PP-1c and iASPP.