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PROJECT PROPOSAL for applicants for ITC fellowships (2017/18) supervisor: institution: contact: CV: Zoltán LIPINSZKI, Ph.D. Institute of Biochemistry [email protected] http://www.brc.hu/file/cv/biochem_lipinszki_zoltan_en.pdf project title: BIOCHEMICAL AND FUNCTIONAL CHARACTERISATION OF PP2A-LIKE PROTEIN PHOSPHATASES IN FRUITFLIES PROJECT SUMMARY Cell division must be carried out with high fidelity in order to maintain genomic integrity. Failure of these processes may lead to genome instability, a hallmark of cancer cells. Reversible protein phosphorylation is one of the highly conserved cellular mechanisms that regulate cell cycle progression. Although roles of protein kinases responsible for phosphorylation are largely understood, the functions of the protein phosphatases that oppose these events are for the most part mysterious. Using genetic, cell biological, biochemical and proteomic approaches we aim to investigate the function of a poorly characterised PP2A-like protein phosphatase, e.g. PP4-like, in Drosophila melanogaster. BACKGROUND The regulatory function of PP2A, PP4 and PP6, the three well established members of the PP2A-type family of Ser/Thr phosphoprotein phosphatases has been linked to various cellular processes, including DNA repair and cell division. Not surprisingly, the dysfunction of these enzymes has already been associated with the progression of human diseases, including cancer. They all function as a heterotrimeric complex consisting of one evolutionarily conserved catalytic (“C”) subunit that associates with a structural protein and one of the several regulatory subunits conferring substrate selectivity and subcellular localisation of the holoenzyme. Although the C subunits of the PP2A-type phosphatases show a high level of amino acid sequence identity, they associate with different set of regulatory proteins, which define their distinct and specific roles in the cell. Recent studies have revealed other members of the PP2A family, whose function has remained elusive. However, in order to understand the complicated phosphorylationdriven regulatory events and the consequences of their dysfunction, characterisation of the poorly known members of the PP2A-type phosphatases must also be carried out. CURRENT RESEARCH We are investigating the mitotic functions of PP4 using Drosophila melanogaster as a model organism. We have recently shown that besides its known centrosomal function, PP4 is necessary to maintain the integrity of the centromere and kinetochore during mitosis. This function of PP4 is independent from other PP2A-type of phosphatases studied so far. An intriguing question, however, has risen by the finding that the gene encoding the catalytic subunit of PP4 (pp4c) has a paralog in Drosophila melanogaster (CG11597), most probably derived from pp4c. CG11597 encodes a PP4C-like catalytic subunit sharing 70 % sequence similarity with the canonical PP4C (including the catalytic triad). Originally, it has been considered as a testis-specific gene suggesting its implication in spermatogenesis (e.g. in meiotic division), which process is broadly studied in different species due to male fertility problems. However, its expression during all stages of ontogenesis in flies has also been reported, which may suggest its more diverged function in other cellular processes as well. Surprisingly, we have never identified PP4C-like in any of our PP4-related proteomic studies, which led us to speculate that PP4Clike may have its own regulatory subunit set that are different for that of other PP4 subunits. According to the current model the carboxymethylation of the conserved Leu307 of PP4C (which is conserved in all PP2A-type catalytic subunits) by LCMT-1 is the prerequisite for the assembly of the heterotrimeric holoenzyme. Interestingly, this Leu is replaced with Gln in PP4C-like strongly suggesting that its regulatory subunits, if any, associate with the catalytic protein in a carboxymethylation-independent manner, which would be a novel type of complex assembly. SPECIFIC AIMS The major aim of this project is to characterise the subunit composition and specific functions of a PP4-like phosphatase. We aim to generate stable cell lines and transgenic flies expressing GFP-tagged PP4C-like, which will be used in cell biological studies as well as in affinity purification of PP4C-like-containing complexes for subsequent mass spectrometric analysis. This would allow us to identify the regulatory subunits and putative substrates of this phosphatase. By means of generating PP4Clike-specific antibody we would be able to test the ontogenetic profile of the protein and its subcellular localization in cultured cells or specialized tissues (e.g. spermatocytes). Using RNAi-based knock down of PP4C-like or replacing the endogenous with a catalytically inactive form of the protein we aim to monitor the consequences of the loss of the enzyme or its activity on mitosis and meiosis. Finally we propose to perform biochemical purification of the PP4-like holoenzyme from flies or reconstitute the complex from recombinant proteins for in vitro dephosphorylation and structural studies. METHODS TO BE LEARNED / APPLIED Establishing stably transfected Drosophila cultured cells and transgenic flies Affinity purification of protein complexes for proteomic studies. Recombinant protein expression and purification in E.coli. Biochemical purification of endogenous protein Classical cell and molecular biological techniques. SUGGESTED READINGS Barr FA, et al.: Protein phosphatases and the regulation of mitosis. J Cell Sci, 124:2323-2334(2011) Chen F, et al.: Multiple protein phosphatases are required for mitosis in Drosophila. Curr Biol, 17:293-303(2007) Berndt N: Serine/threonine-specific protein phosphatases and cancer. Exp Opin Therap Targets, 4:581608(2010) Helps ND, et al.: Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos. J Cell Sci, 111:1331-1340(1998) Adam C, et al.: Conservation of male-specific expression of novel phosphoprotein phosphatases in Drosophila. Dev Genes Evol, 220:123-128(2010) SNAPSHOTS FROM THE HOST LABORATORY Significant publications Fu j, Lipinszki Z, et. al.: Conserved molecular interactions in centriole-to-centrosome conversion. Nature cell biology. 18:87-99.(2016) Lipinszki Z, et al.: Centromeric binding and activity of Protein Phosphatase 4. Nat. Commun., 6:5894(2015) Lipinszki Z, et al.: Affinity purification of protein complexes from Drosophila embryos in cell cycle studies. Meth Mol Biol, 1170:571-88(2014) Lipinszki Z, et al.: A novel interplay between the ubiquitin–proteasome system and serine proteases during Drosophila development. Biochem J, 454:571-83(2013) Lipinszki Z, et al.: Ubiquitylation of Drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors. Biochemistry, 51:2461-70(2012) Representative recent research grants Currently our research is supported by 3 research grants: OTKA-PD 115404, GINOP-2.3.2-15-2016-00001, GINOP2.3.2-15-2016-00032 Supported by the TÁMOP 4.1.1.C -13/1/KONV.2014-0001 project