Evaluation of the substrate specificity of several protein tyrosine phosphatases involved in diseases

Evaluation of the substrate specificity of several protein tyrosine phosphatases involved in diseases 2008 – 2010
Budget: Total 2.000.000 lei/ Cost for IBAR: 1.700.000 lei
Project director: Stefan Szedlacsek
PNCDI-II-04-3452, (no:41-038/2007) - Program 4 - PARTNERSHIP IN THE AREAS OF PRIORITY

Protein tyrosine phosphatases (PTPs) are fundamental regulatory enzymes that dephosphorylate phosphotyrosine residues and are essential components of intracellular signalling pathways in both normal and pathological conditions. There is experimental evidence that disruption of the phosphatase activity of many PTPases is involved in the pathogenesis of several congenital or acquired diseases, including diabetes, cancer, infections, autoimmune, neuronal and cardiovascular diseases.

In this project, we propose to study three important human PTPases that play key roles in the human body under both normal and pathological conditions: a) the first one is massively overexpressed in colorectal cancer metastasis; b) the second one is involved in the branchio-oto-renal syndrome (Melnick-Fraser syndrome) and c) the third one is encoded by a gene located on chromosome 18pter-q11, in a region with frequent abnormalities involved in human cancer; it also shows a high homology with the tumour suppressor PTPRT - PTPase, which has the most frequent mutations in colorectal cancer tumours. The elucidation of the molecular mechanisms governing the involvement of PTPases in different diseases is under the sign of some major questions, namely: what is the substrate specificity of these phosphatases, respectively what are their physiological substrates? The aim of this project is to respond to this challenge by applying new experimental techniques. The substrate specificity data obtained will be compared with those obtained by the substrate trapping technique in order to obtain an overall picture of the substrate specificity of the investigated PTPases. The results of these studies will be the starting point for the applied research in our project aimed at realising the sequences of specific inhibitors that can be materialised in antitumour drugs with high specificity and efficiency. The methods described in this project are modern methods that can be adapted to the high-throughput technologies of today's pharmaceutical industry.

Stefan Szedlacsek, Dr.
Stefan Szedlacsek, Dr.

Head of Department

Prof. Dr. Stefan Szedlacsek is the Head of the Enzymology Department at the Institute of Biochemistry of the Romanian Academy. He holds a PhD degree in Biotechnology from Polytechnical University of Bucharest as well as a MSc in Organic Synthesis (Polytechnical University, Bucharest) and MSc in Mathematics (University of Bucharest). As a visiting scientist, he performed research in the field of cholesterol metabolism at the University of Illinois at Urbana-Champaign (USA), where he succeeded to evidence a new pathway in the metabolism of oxysterols.  He is an “Alexander von Humboldt” fellow and worked in Germany, in the Institute of Biochem More...

Project coordinator: Dr.Stefan SZEDLACSEK

Partner 1: Politehnica University of Bucharest     - Project manager: Dr. Mihalea Camelia BĂLAȘU

Partner 2: CORAX - BIONER CEU ROMANIA SA   - Project manager: Prof.Dr. Lániy SZABOLCS





I.  Cloning, expression and purification of two PTPases involved in different human pathological conditions.

II. Investigation of the substrate specificity of the three PTPases studied using the substrate trapping or pull-down technique.

III. Development of new biochemical methods to confirm substrate specificities and potential substrates.

 In the first stage, the cDNA containing the catalytic domain of RPTPμ was cloned into a prokaryotic expression vector, resulting GST-ΔJMD1μ construct. The recombinant construct was expressed in E. coli as a protein fused with GST (glutathione S-transferase). Further on, the protein was purified to high purity using two subsequent chromatographic methods and the resulting protein was used for in vitro dephosphorylation assays and for GST-pull down experiments. Study of PLCγ2 as a potential substrate of RPTPμ was approached by in vitro dephosphorylation experiments. The starting point was represented by the results of a previous experimental study performed on a library of 360 phosphopeptides. According to this study, a tyrosine-phosphorylated tridecapeptide identical in sequence to the sequence surrounding the Y753 residue from the sequence of phospholipase Cγ2 (PLCγ2) was efficiently dephosphorylated by the catalytically active domain of RPTPμ. A fragment of PLCγ2 containing its both SH2 domains and the SH3 domain (SH2-SH2-SH3) was cloned and expressed in a prokaryotic system. Also, the mutated forms of SH2-SH2-SH3 fragment Y753F and Y759F were obtained. Then, the expressed proteins were purified, phosphorylated by Lck kinase and subsequently, submitted to in vitro dephosphorylation by the catalytic domain of RPTPμ. The experimental data thus obtained suggested that both phosphotyrosine residues (pY753 and pY759) have been dephosphorylated by the catalytic domain of RPTPμ, but pY753 was the preferred site.

Also in this stage of the project the sequence of the coding gene of human Eya was amplified by PCR from a human cDNA library (MegaMan Human Transcriptome from Stratagene). The amplified sequence was cloned into pGEX-4T1 and pHAT2 prokaryotic expression vectors.

In the second stage the evaluation of substrate specificity was performed. In the same time, the identification of native substrates of RPTPµ, hEya and hPRL3 was tried using substrate trapping and GST-pull down techniques. Substrate trapping consist in catching native substrates from various cellular extracts taking use of inactive mutants of PTPs which still have the ability to bind substrates. First, the inactive trapping mutants (D/A and C/S) were obtained for all PTPs by site direct mutagenesis. For RPTPµ, D1063A and C1095S mutants were obtained for the GST-ΔJMD1µ. Then each mutant was expressed in E. coli as GST-fusion proteins and finally the purification was performed with specific chromatographic methods. Thus, proteins with high purity were obtained and were used in substrate specificities studies. For RPTPµ both substrate trapping mutants were used in GSD pull-down experiments to test the interaction of RPTPµ with its potential substrate PLCγ2.

The binding potential of immobilized of RPTPµ trapping mutants was tested on GSH-Sepharose B both for SH2-SH2-SH3 fragment and the integral PLCγ2. PLCγ2 integral form was obtained by transfection in mammalian cells and phosphorylation using inhibition of endogenous cellular phosphatases. Results demonstrated that both substrate trapping mutants were able to trap the SH2-SH2-SH3 and the integral from PLC γ2 . D/A mutant proved to be most efficient one in substrate trapping. 

To evaluate the substrate specificity of Eya protein the substrate trapping mutants were obtained. To this purpose the first aspartate from DXDX(T/V) motif I was substituted to either alanine (A) or asparagine (N). During the first two stages of the project we optimized the expression for wild-type and mutant Eyes absent genes and further on we optimized the purification methods for these proteins.

As concerning the pull-down of PRL3 substrates, we first obtained the catalytic inactive trapping mutants D72A and C104S, knowing that the residues C104 from the phosphate binding loop and respectively D72 from the WPFDD loop are essential for the PRL-3 catalytic activity. Mutant proteins were first obtained as N-terminal polyhistidine-tagged recombinant proteins (6xHis). Subsequently, the PRL3-wt and inactive mutant’s genes were subcloned into pGEX-4T1 vector. The optimal conditions for expression in prokaryotic system and protein purification of PRL3 were established.

            Within stage three, confirmation of substrate specificity of RPTPµ for PLCγ2 was realized, using for this purpose a new technique based on use of sulfotyrosine derivative of SH2-SH2-SH3 fragment. Tyrosine sulfated proteins were obtained using the method reported by Peter Schultz and his team from the Scripps Research Institute (11). The method consists in using aminoacyl-tRNA synthetase (aaRS) from Methanococcus janaschii, which is able to specifically load sulfotyrosine on a suppressor tRNA of the amber stop codon (TAC). When orthogonal pair tRNA/aarS is expressed in E. coli sulfotyrosine is inserted in proteins at positions corresponding to the amber codon. The following strategy was used: the codon of tyrosine753 was substituted for amber codon (TAC) by site directed mutagenesis. Than, the vector containing the SH2-SH2-SH3 fragment with Y753 mutated to stop codon was coexpressed in E .coli together with the plasmid coding for tRNA/aaRS, in a minimal medium containing sulfotyrosine. A set of expression parameters (temperature, medium composition, sulfotyrosine concentration, IPTG concentration and induction time) were tested, eventually leading to optimal expression condition of SH2-SH2-SH3 containing sulfo-Y753. The sulfotyrosine containing protein was further purified by affinity chromatography. Preparation of sulfotyrosine containing recombinant protein is a remarkable achievement given that initiate new methods of study for substrate specificity of PTPases. The sulfotyrosine used in expression of proteins was obtained by Partner I- The research group of Department of Organic Chemistry (Polytechnic University, Bucharest). Synthesis of sulfotyrosine (which is not commercial available) was performed using a procedure based on the esterification of -OH group of L-tyrosine with chlorosulfonic acid and separation of tyrosine sulfate as a sodium salt, following ester precipitation with ethylic ether (average conversion of tyrosine: 85%). The preparation thus obtained by chemical synthesis was tested by TLC and results indicated the presents of the tyrosine sulfate. These experimental data were also in agreement with the NMR and IR spectra. 

The evaluation of substrate specificity of human Eyes absent protein was performed using a phosphopeptide microarray screening based on 6200 phosphotyrosine-containing peptides derived from tyrosine phosphorylated human proteins. The results indicated that Eya phosphatase completely dephosphorylate 7 phosphotyrosine-containing peptides, suggesting that at least one of the proteins could be a potential substrate. To test this hypothesis, we synthesized AHDGGI-pTyr-AISWSP phosphopeptide, which correspond to WDR1 protein (actin-interacting protein-1) and used it in dephosphorylation reaction catalyzed by Eya phosphatase. The in vitro tests revealed that the phosphopeptide derived from WDR1 is dephosphorylated by Eya protein, being the first evidence that this protein can be a potential substrate for Eya. To test whether the full-length WDR1 is dephosphorylated by Eya, initially we obtained a lysate of HEK293T which contained phosphorylated proteins (including overexpressed phosphorylated WDR1). Phosphorylated proteins from lysate were subjected to dephosphorylation in presence of hEya phosphatase (hEya-wt and hEya-D/N) and the reaction mixture was than analyzed by western blot using specific anti-phosphotyrosine antibodies. In this way we observed that a protein, with the molecular weight of about 90kDa corresponding to the (molecular weight predicted for the fusion protein YFP-WDR1) was specifically dephosphorylated by hEya-wt. The result is to be confirmed using specific antibodies against anti-WDR1 or anti-YFP.   

            The present project had also an applied objective towards development of potential antitumoral therapies. To this purpose Partner 2 - Sc CORAX-Bioner-Romania S.A. - performed molecular docking studies for a peptide derived from a native substrate of PRL3 (ezrin) as well as for two inhibitors of PTPases. Within docking studies the enzyme-substrate interactions were analyzed using two reported structures of PRL.

The results of the complex research studies obtained within the present project highlighted new findings regarding the evaluation of the substrate specificities of the analyzed PTPases. Identification of the new native substrate of RPTPµ (PLCγ2) has a significant value in understanding the physiological role played by RPTPµ in cellular signaling.  In addition, our results indicated WDR1 as a potential substrate for Eya.

An essential tool in finding new procedures for the study of substrate specificity of PTPases is represented by the use of the proteins which contain sulfotyrosine as a non-hydrolysable analog of phosphotyrosine. Thus, preparation in this project of a protein fragment derived from PLCγ2 in which sulfotyrosine substitutes for phosphotyrosine represents an important starting point for setting up this new type of procedures.

Attending conferences, congresses;

The Annual International Conference of RSBMB Bucharest:

(1) Identification of an in vitro substrate of protein tyrosine phosphatase RPTPµ”; Authors: Rodica Badea, Stefan E. Szedlacsek; Rom. J. Biochem., 45(1), 2008, p.51/ISSN 1582-3318/Ed.Academiei Romane

(2) Enzymatic Characterization of a New Isoform of Human Eyes Absent Homolog 3”; Authors: Mihaela Pascaru, Stefan E. Szedlacsek; Rom. J. Biochem., 45(1), 2008, p.60/ISSN 1582-3318/Ed.Academiei Romane

 FASEB summer research conferences “Protein Phosphatases”, July, 13-18, 2008; Snowmass, Colorado

 (1) Analysis of molecular determinants of PRL-3”; Authors: Mihaela Pascaru, Carmen Tanase, Andrei M. Vacaru, Elena Neagu, Patricia Boieti, Irinel Popescu,  Stefan E. Szedlacsek

(2) Identification of an In Vitro substrate of receptor protein tyrosine phosphatase RPTPµ"; Authors: Rodica Badea, Stefan E. Szedlacsek

 Congress of the International Union of Crystallography, August, 23-31, 2008; Osaka, Japan

(1) „Structure-function analysis of Eyes absent proteins - aspartate dependent protein tyrosine phosphatases”; Authors: Mihaela Pascaru, Stefan E. Szedlacsek

 EMBO Conference “Europhosphatase 2009” on Protein phosphatases in development and disease:

(1) ”Interface Analysis of the Complex between ERK2 and PTP-SL”; Authors: M. Bălaşu, L. Spiridon, S. Miron, C. Crăescu, A. Scheidig, A-J Petrescu ; S. Szedlacsek July 14-19th, Egmond aan Zee, (The Netherlands);

(2) ”New evidence for PLCγ2 as a potential substrate of RPTPμ”; Authors: Rodica Badea, Mihaela Menţel (Pascaru), Stefan Szedlacsek


"Analysis of molecular determinants of PRL-3"; Authors: Pascaru M, Tanase C, Vacaru AM, Boieti P, Neagu E, Popescu I, Szedlacsek SE; J Cell Mol Med ISSN 1582-1838/DOI.10.1111/j.1582-4934.2008. 00591.x, ED. WILEY -BLACKWELL