Download PROJECT PROPOSAL for applicants for ITC fellowships

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