Exploring the role of nuclear phosphatases and their associated protein in cell growth and development

Duncalf, Louise Exploring the role of nuclear phosphatases and their associated protein in cell growth and development. PhD thesis, University of Liverpool.

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Abstract

Reversible protein phosphorylation is a key regulatory mechanism for controlling various cellular processes that determine cell fate. Phosphorylation status is controlled by kinases and phosphatases, which phosphorylate and dephosphorylate specific residues in target proteins respectively. Protein Phosphatase 1 (PP1) is a major serine/threonine phosphatase that is highly conserved in eukaryotes. Its catalytic subunit (PP1c) associates with numerous subunits that target and regulate its activity to specific subcellular localisations and substrates to define its role in various processes. PP1 Nuclear Targeting Subunit (PNUTS) is one of the most abundant regulatory subunits of PP1 in the nucleus. The aim of this work was to characterise a Drosophila orthologue of PNUTS and identify interacting proteins to further understand the role of PNUTS-PP1 in nuclear processes using various genetic and biochemical approaches. Mutational analysis revealed dPNUTS is essential for cell growth and survival as mutant animals die as 1st instar larvae and mutant cells are eliminated from developing tissues. PNUTS-PP1 co-localise on Drosophila polytene chromosomes, with many sites also marked by RNA Polymerase II (RNAPII). Biochemical analysis revealed Serine 5 of the C-terminal domain of RNAPII (CTD-Ser5) is a possible substrate of PP1-dPNUTS. Various RNAPII-dependent genes are misregulated in dPNUTS mutant animals, including genes involved in metabolic processes, most likely as a consequence of deregulated CTD-Ser5 phosphorylation. Another possible mechanism could be through regulation of histone modifications that determine gene expression patterns since clonal analysis revealed various histone marks are upregulated in dPNUTS and PP1 mutant cells. Methodologies to screen for regulatory enzymes affecting histone phosphorylation were also assessed. To further understand the role of dPNUTS in cell growth and development, a yeast two-hybrid approach together with biochemical analysis was used to identify dPNUTS interacting proteins. The top hit was dTOX4, the Drosophila homologue of LCP1/TOX4, which binds to PNUTS in humans. Mutational analysis revealed dTOX4 is essential for fertility in female and male adult flies. Homozygous mutant females displayed defects in nurse cell chromosome dispersal and abnormal dorsal-ventral patterning in the oocyte. Homozygous mutant males failed to complete spermatogenesis and exhibited a range of abnormal phenotypes in the testes, including defective cytokinesis, small nuclei, decondensed chromatin and Stellate crystal formation. Novel dPNUTS-interactors were also identified, which could offer further mechanistic insight into dPNUTS-PP1 function.

Item Type:	Thesis (PhD)
Additional Information:	Date: 2014-12-18 (completed)
Subjects:	?? Q1 ??
Depositing User:	Symplectic Admin
Date Deposited:	27 Aug 2015 09:33
Last Modified:	17 Dec 2022 01:38
DOI:	10.17638/02012842
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2012842