Zhou, Y; (2014) Developmental Functions of Drosophila ASPP and RASSF8. Doctoral thesis , UCL (University College London).

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Abstract

Epithelial cells are connected to each other via intercellular junctions, which are established, remodelled and maintained by a complex molecular machinery. Aberrant regulation of cell-cell junctions leads to loss of tissue organisation and is a hallmark of cancer formation. Although many kinases that regulate the phosphorylation of junctional proteins have been described, much less is known about the reversal of phosphorylation by phosphatases. This thesis investigates the role of Drosophila ASPP, a scaffold protein that is localised at adherens junctions, as a regulatory subunit for PP1s. In vitro, ASPP can bind to PP1 using its RVXF motif and SH3 domain. In vivo, ASPP co-localises with the PP1a96A and PP1b9C isoforms at cell-cell junctions in the pupal retina and ASPP function is at least partially dependent on its ability to bind to PP1. Furthermore, ASPP can recruit two additional coiled coil containing scaffold proteins, RASSF8 and Ccdc85, to form trimeric complexes with PP1a96A. ccdc85 mutants that were generated in this work have a rough eye, similar to ASPP mutants, suggesting a similar function in vivo. Two potential substrates for the ASPP/PP1 complex were tested: (1) Yki a transcriptional co-activator that is part of the Hippo pathway and (2) Baz, a scaffold protein that is required for cell polarity. Although no evidence for dephosphorylation of Yki was found, Baz can be dephosphorylated in vitro. The well-described aPKC phosphorylation site (S980) and five additional serine/threonine residues are strongly dephosphorylated by ASPP/PP1. My work also identified three potential regulators/scaffolds of ASPP/PP1. The Hippo pathway kinase Wts can phosphorylate RASSF8, the E3 ubiquitin ligase Sina can ubiquitylate and degrade ASPP and the junctional protein Magi can associate with RASSF8 at adherens junctions. Finally, novel potential regulators, scaffolds or substrates of the ASPP/PP1 complex were identified through AP-MS experiments and tested for their ability to modulate the ASPP depletion phenotype in vivo.

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