PP2A is a heterotrimer that consists of a catalytic subunit (PP2A-C) whose activity, substrate specificity and localization is regulated by the binding of a scaffolding A (PP2A-A) and a regulatory B (PP2A-B) subunits. Recent discoveries have showed the role of PP2A as tumor suppressors and in resistance to platinum-based antitumoral agents in mammals and in Alzheimer disease in humans. The project will promote the exchange of ideas and the execution of joint experiments taking advantage of the scientific tools and resources independently developed in the partner laboratories. The expertise of the Project Promoter research group on PP2A-C will be combined with the expertise of the donor partner, University of Stavanger, PP2A-B for elucidating the manifold involvement of PP2A in plant signalling, aiming to understand the molecular responses to environmental challenges. The project is expected to benefit the laboratories and institutions involved and the scientific community in general.
Summary of project results
Reversible protein phosphorylation is a common mechanism in both prokaryotic and eukaryotic transduction pathways to connect external and/or internal signals to a given cell response. The phosphorylation status of the target protein depends on the balance of the opposing kinase and phosphatase activities. Type 2A serine/threonine phosphatases (PP2A) are well conserved in eukaryotes, in both sequence and functional properties. PP2A is a heterotrimer that consists of a catalytic subunit (PP2A-C) whose activity, substrate specificity and localization is regulated by the binding of a scaffolding A (PP2A-A) and a regulatory B (PP2A-B) subunits. The Arabidopsis genome codes for five PP2A-C, three A, and 17 B, B’, and B’’ subunits (Farkas et al., 2007), theoretically accounting for 255 putative heterotrimers that eventually could perform differently. Identifying PP2A activities in specific signalling pathways, and understanding how specificity is achieved through subtle changes in the composition of the heterotrimer, is therefore a key knowledge for breeding strategies towards an improved, sustainable crop production. This work more specifically addressed the role of PP2A in nitrogen assimilation, and carbon/nitrogen balance, pivotal parameters for sustainable crop production. The specific goals and activities performed of the project were the following: 1) Establishing the subcellular localisation of PP2A-B and –C subunits. 2) Analysing the nitrate reductase activity in PP2A-C mutants. 3) Analysing PP2A involvement in environmental signalling 4) Characterizing the interaction of PP2A-B and –C subunits. 5) Identifying PP2A target proteins by phosphoproteomic analysis The results obtained increase our understanding of the molecular mechanisms endowing specificity to protein phosphatase 2A, thus allowing its participation in a myriad of signaling networks. Due to the high conservation of protein phosphatase 2A throughout eukaryotes, the generated knowledge is relevant for a better understanding of regulatory signaling pathways involved in fundamental processes. The recent discoveries on the role of PP2A as tumor suppressors and in resistance to platinum-based antitumoral agents in mammals, and in Alzheimer disease in humans have put a strong momentum to this study. A publication was in preparation at the closure of the NILS funding, summarizing the results obtained on the involvement of PP2A-mediated dephosphorylation in the regulation of NR activity.
Summary of bilateral results
The project has allowed the coordination of activities between the research groups at the National Centre of Biotechnology CSIC in Spain and the University of Stavanger in Norway, working in a closely related, complementary subject, namely the role of protein phosphatase 2A in the development of the model plant Arabidopsis thaliana. Both partners provided different complementary expertise. National Centre of Biotechnology CNB-CSIC in Spain has generated the Arabidopsis pp2a-c double mutant collection and the expertise in yeast two-hybrid- techniques. Monitoring NR activity is a routine technique at the University of Stavanger. On May 2015, partners submitted a project application entitled “Function of Protein Phosphatase 4 and Environmental Cues” to the FRIMEDBIO (TOPPforsk) programme of the Norwegian Research Council, leaded by Catherine Lillo from University of Stavanger. The CNB-CSIC laboratory participates in the project as main international partner.