Group 6 : Phosphatase team (E3) PI : Alphonse GARCIA

 
1. Team members.
Created in 2008, the E3 phosphatase team is presently defined by two full time researchers (A.Garcia and P.Falanga) and a PHD student (A.Galioot-thesis in October 2013)
 
2. Title of the research
PP1c and PP2A1 protein phosphatases are key cell regulators and powerful therapeutic targets
 
3. Scientific background
General scientific background
            The PP1/PP2A family of serine/threonine protein phosphatases has been implicated in the regulation of many cellular and infectious processes including apoptosis in mammalian cells (Garcia et al.comprise dimeric or heterotrimeric enzymes. The dimeric PP2A core enzyme consists of a catalytic C subunit (PP2Ac) and a structural A subunit. A third subunit (B) can also bind to the core and regulate both the substrate specificity and localization of trimeric PP2A holoenzymes. 2000a). PP1 and PP2A proteins do not exist as free catalytic subunits in the cell. PP1 proteins are oligomeric complexes comprising a catalytic structure (PP1c), exerting enzymatic activity, associated with an interacting subunit (Garcia et al. 2003). PP2A proteins
 
Specific scientific background including salient concepts
            PP1 signature
We have initially characterized in the BH1 and BH3 domains of some Bcl-2 proteins two distinct functional PP1 consensus docking motifs, R/Kx(0,1)V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death regulation (Ayllon et al.http://pp1signature.pasteur.fr 2000). In this regard, we have also further proposed a concept called PP1-signature based on bioinformatic identification of peptide sequences surrounding PP1c docking sites in important regulatory target proteins. To develop this potentiality, we have created a new web site: that allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library (for details see Garcia et al. 2004).
            PP1 apoptotic signature
We recently published that the combinatorial PP1c docking motif R/Kx(0,1)V/IxFxxR/KxR/K, deduced from AIF562-571 and APAF-1122-131 sequences, is a new PP1c-dependent Apoptotic Signature (Godet et al. 2010a). We have proposed to use this motif as a new tool for drug design to characterize new potential anti-tumor molecules.
            Drug Phosphatase Technology (DPT)
To specifically deregulate intra-cellular pathways, we previously validated and published an original approach, named Drug Phosphatase Technology (DPT) (Guergnon et al.illustrated in Fig.1. This approach is based on the design of peptides containing sequences allowing intra cellular delivery (cell permeable peptides, cpp) and specific interaction with PP1/PP2A holoenzymes in order to disrupt or modulate specifically phosphatase-mediated pathways. We have characterized a novel cell penetrating peptide, the DPT-sh1 shuttle, capable of penetrating several cell lines, chick embryos and human red blood cells infected with the malaria parasite P. falciparum. Intracellular delivery by DPT-sh1 of PP1/PP2A docking sites, respectively from PP1-interacting domain of Bad or from PP2A-interacting domain of CD28 antigen, provokes cell death. 2006)
 
 
 Figure 1: Drug Phosphatase Technology : a concept to interfere with PP1/PP2A-mediated normal and pathological pathways
           
PP2A a key target for viruses
 
 
Several viruses encode small proteins that can directly interact with cellular PP2A holoenzymes in order to control the major PP2A-dependent regulatory pathways of their hosts (Guergnon et al.The trimeric ABaC holoenzyme, named PP2A1, represents a key target selected by some viruses to take the control of infected cells (Fig.2). Small t antigens of papovaviruses are viral proteins able to replace the cellular Ba subunit within PP2A1 holoenzyme. Small t can deregulate the Ras pathway, induce transcription and cell proliferation (Sontag et al.In contrast to small t, the interaction of adenoviral protein E4orf4 with the regulatory Ba subunit of trimeric PP2A1 specifically induces a p53-independent apoptotic process in infected and tumor cells (review in Guergnon et al.. 2011) 1997; Garcia et al. 2000b). 2000a).
More recently, we found that the C-terminal flexible domain of Vpr protein from HIV-1 strain 89.6 can bind to the structural A subunit of PP2A1 holoenzyme to induce apoptosis in human cell lines (Godet et al. 2010b).
 
 
 
 
 Figure 2: PP2A: a target for viral proteins
 
 
4. Research projects
 
Goals: The E3 phosphatase group is presently mainly focused on two aspects
            a-to study PP2A mediated regulatory pathways involved in survival of tumor cells (neuroblastomas and gliomas) and tau-phosphorylation.
            b-to study PP1/PP2A mediated regulatory pathways involved in regulation of spinal muscular atrophy (SMA) (Coll. Suzie-Lefèvre, INSERM).
 
Main results:
            We have identified three families of DPT-peptides (3 to 10 members) containing short PP2A interacting sequences. These domains were deduced from cellular caspase-9, DPT-C9 family (Coll. Xavier Cayla, INRA), adenoviral E4orf4 (DPT-E4 family) and HIV-1 Vpr (DPT-Vpr family).
            We identified a functional polymorphism in the C-Terminal domain of HIV-Vpr (Coll. JH Colle, Institut Pasteur).
            We have identified a family of DPT-peptides (3 members) containing short PP1 interacting sequences deduced from cellular Gemin8 (Coll. Suzie Lefèvre, INSERM).
 
Main objectives
            1-Structure function analyses of DPT-E4 family members of peptides mimicking E4orf4-mediated anti-cancer pathways: A.Galioot /A.Garcia (Coll. P.England and B. Baron, Institut Pasteur).
            2- characterization of the effects of PP2A-interacting DPT-peptides on survival and tau phosphorylation of human neuroblastomas: A.Galioot-A.Garcia (Coll. Luc Buée INSERM).
            3- analysis of the effects of DPT-peptides on PP2A-mediated radio-sensitization pathways in glioma cells. PB Falanga (Coll. Marie Dutreix, Institut Curie and Solange Lavielle, UPMC-ENS).
            4- effects of PP2A-interacting DPT-peptides on senescent pathways in gliomas: A.Garcia (Coll. Brigitte David-Watine IP).
            5- characterization of potential DPT-therapeutic molecules against spinal muscular atrophy (SMA): A.Garcia  and PB Falanga (Coll. Suzie-Lefèvre, INSERM).
 
5. Main technical know-how:
Cellular biology including Protein-phosphatases, apoptosis and radio-sensitivity analyses.
 
6. References
Ayllón V, Martinez A.C, Garcia A, Cayla X, and Rebollo A. Protein phophatase 1-a is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. (2000) EMBO J. 19: 1-10.
Garcia A, Cayla X, Sontag E. Protein Phosphatase 2A : a definite player in Viral and Parasitic regulation. (2000a) Microbes. Inf. 2: 401-407.
Garcia A, Cereghini S, Sontag E. Protein Phosphatase 2A and Phosphatidylinositol 3-kinase regulate the activity of Sp1 responsive promoters (2000b) J.Biol.Chem. 275: 9385-9389
Garcia A, Cayla X, Guergnon J, Dessauge F, Hospital V, Rebollo MP, Fleischer A, Rebollo A. Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. (2003) Biochimie. 85 :721-726.
Garcia A, Cayla X, Ayllon V, Deveaux E, Caudron B, Rebollo, A. New insights in protein Phosphorylation: Two distinct motifs define a signature for Protein phosphatase 1 interacting proteins. (2004) C.R. Biologies 327 : 93-97.
Godet A N, Guergnon J, Maire V, Croset A, Garcia.The combinatorial PP1-binding consensus motif (R/K)x (0,1)V/IxFxx(R/K)x(R/K) is a new apoptotic signature. PloS ONE (2010a) 5: e9981.
Godet A N, Guergnon J, Croset A, Cayla X , Falanga PB, Colle JH, Garcia. PP2A1 binding, cell transducing and apoptotic properties of Vpr77-92 a new functional domain of HIV-1 Vpr proteins PloS ONE (2010b) 5: e13760.
Guergnon J, Dessauge F, Dominguez V, Viallet J, Cayla X, Rebollo A, Yuste V, Susin S, PE. Bost PE, and Garcia A. Use of penetrating peptides interacting with PP1/PP2A proteins as a basis for a new Drug Phosphatase Technology. (2006) Mol Pharmacol, 69 :1115-1124.
Guergnon J, Godet Angélique N, Galioot Amandine, Falanga Pierre Barthélemy, Colle Jean-Hervé, Cayla Xavier, Garcia Alphonse. “PP2A targeting by viral proteins : a widespread biological strategy from DNA/RNA tumor viruses to HIV-1” Biochimica et Biophysica Acta 1812 (2011) 1498–1507.
Sontag E, Sontag , Garcia A. Protein Phosphatase 2A is a critical regulator of protein kinase C z signaling targeted by SV40 small t to promote cell growth and NF-kB activation. (1997) EMBO.J. 16: 5662-5671.
 
 
 
 
Team members
 
 

Alphonse Garcia
Chef de Laboratoire,
Responsable du Groupe E3-Phosphatase
Institut PasteurTel/ 01 40 61 38 21
email: agarcia@pasteur.fr
 
 

Pierre FALANGA
Groupe E3-Phosphatases
Chargé de recherche IP
Tel. 01 40 61 38 16
Email : pierre.falanga@pasteur.fr



 
Amandine GALIOOT
Étudiante 3ème année de thèse
Laboratoire E3-Phosphatases
01 40 61 38 16
amandine.galioot@pasteur.fr