Only 30 serine/threonine phosphatases balance the action of more than 400 serine/threonine kinases in human cells. This disproportion is compensated by the ability of major serine/threonine phosphatases (e.g. PP1 and PP2A) to perform different functions. A modular assembly where an invariant catalytic subunit couples to different regulatory subunits contributes to the flexibility: different compositions may be associated to different functions. We recently reported that a PP2A regulatory subunit (PP2A Bb) is expressed by T cells only during low IL-2 conditions. The expression of this molecule is associated to apoptosis induction: its over- expression triggers apoptosis while its silencing inhibits it during IL-2 deprivation. Based on that, we have proposed that PP2A Bb regulates apoptosis triggered by IL-2 deprivation by conferring PP2A the capacity to induce apoptosis. This novel concept, that a specific PP2A B subunit is linked to a specific function of PP2A and that such function can be regulated through the control of the individual B subunit, represents a step forward in the understanding of the complex physiology of serine/threonine phosphatases. The first part of this project is aimed at identifying which cells depend on PP2A Bb for apoptosis induction and understanding how IL-2 deprivation induces PP2A Bb upregulation. The second part will explore the apoptosis- inducing pathways triggered by PP2A Bb. The experiments will be performed in primary human T cells. We will detect PP2A Bb expression by western blot and real time PCR. To analyze its transcriptional regulation, we have developed a plasmid that contains the promoter region of the gene that codes for PP2A Bb coupled to a fluorescent protein. This allows us to image the cells actively transcribing PP2A Bb at a single cell level using confocal microscopy and flow cytometry. Analyses of the pathways utilized by PP2A Bb to induce apoptosis will be mostly studied at the protein level. Activation of caspases and Bcl-2-related proteins along with mitochondrial integrity will be evaluated. For this, we will rely on over-expression and silencing systems used by us previously. At the conclusion of the project, we will know which T cell subsets are deleted in a PP2A Bb-dependent manner and which elements of the T cell activation process confer susceptibility to this type of apoptosis. We will understand the transcription factors involved in the regulation of PP2A Bb expression and the cellular mechanisms involved in PP2A Bb-triggered cell death. This information will be relevant for increasing the knowledge of a process involved in autoimmune and lymphoproliferative diseases. Moreover, it represents a conceptual breakthrough in our understanding of the regulation of PP2A in a discrete function.
This project will study one function of a multi-tasking human enzyme: the regulation of death of T lymphocytes. It is important since it will show us how the enzyme PP2A is regulated and what factors determine the fate of lymphocytes after an immune response. It is essential to understand these processes since ill regulation of PP2A and cell death causes autoimmune disease and cancer.
|Rodríguez-Rodríguez, Noé; Apostolidis, Sokratis A; Fitzgerald, Lauren et al. (2016) Pro-inflammatory self-reactive T cells are found within murine TCR-??(+) CD4(-) CD8(-) PD-1(+) cells. Eur J Immunol 46:1383-91|
|Rodríguez-Rodríguez, Noé; Apostolidis, Sokratis A; Penaloza-MacMaster, Pablo et al. (2015) Programmed cell death 1 and Helios distinguish TCR-??+ double-negative (CD4-CD8-) T cells that derive from self-reactive CD8 T cells. J Immunol 194:4207-14|