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| Author(s): |
Ana Amélia Sanchez Iacia
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2021-05-25 |
| Examining board members: |
Juliana Helena Costa Smetana;
Daniel Maragno Trindade;
Luis Antonio Peroni;
Matheus de Castro Fonseca;
Fábio Cesar Gozzo
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| Advisor: | Juliana Helena Costa Smetana |
| Abstract | |
TIPRL (TOR Signaling Pathway Regulator-like) is a regulatory protein that interacts with the catalytic subunits of type 2A phosphatases (PP2A, PP4 and PP6), a set of phosphoserine and phosphothreonine phosphatases that accounts for a large part of intracellular dephosphorylation events. These phosphatases regulate several cellular processes, such as apoptosis, DNA damage response and cell division, and PP2A is an important tumor suppressor, negatively regulating several oncogenic signaling pathways. TIPRL is overexpressed in several human carcinomas and possibly exerts a pro-tumor function through the inhibition of PP2A by mechanisms that are still poorly understood. TIPRL presents a unique folding with a conserved cleft which binds the conserved carboxyterminal tail of the catalytic subunit of PP2A (PP2Ac) and presumably also of PP4c and PP6c that show sequence similarity with PP2Ac in that region. This tail harbors two phosphorylation sites (T304 and T307) and one carboxymethylation site (L309), where it undergoes reversible post-translational modifications that regulate the selective recruitment of regulatory subunits. Phosphorylation inhibits PP2Ac and impairs the formation of the active holoenzyme, which is composed of a scaffold subunit (PR65/A) and a subset of regulatory subunits (B, B', B" and striatins). Methylation is necessary for the recruitment of some B-type subunits. TIPRL inhibits PP2A activity in vitro, but the mechanism for PP2A regulation by TIPRL in the cell has not yet been elucidated. More specifically, the functional consequence of these modifications in the PP2Ac interaction with TIPRL is still unknown. In this study, we examined the interaction between PP2Ac and TIPRL in cell lines using stable (NIH-3T3 and N2a), or transient expression (HEK293T), of phosphomimetic mutants and their respective non-phosphorylable counterparts, as well as deletion of the C-terminal leucine which prevents carboxymethylation. In immunoprecipitation and Western blot analyses, we observed that the TIPRL interaction is extremely sensitive to mutations in the C-terminal of PP2Ac, and also to pH. The T304D phosphomimetic mutation significantly increased the TIPRL interaction with PP2Ac, whereas Y307F, Y307E and L309? mutations completely abolished the interaction. TIPRL interacted exclusively with the non-methylated version of PP2Ac. TIPRL overexpression decreased the interaction of PP2Ac with B subunit in transiently transfected cells, but this result did not reproduce in stable cell lines. The interactions of TIPRL mutants with PP2Ac, PP4c and PP6c were also analyzed. These mutants were selected for their potential to abolish the interaction with the PP2Ac tail, however, some of them surprisingly have gained interaction with PP4c and PP6c. The stable lines developed in this study allowed us to analyze the TIPRL - PP2Ac interaction in a semi-quantitative and reproducible way. The preference of TIPRL for the non-methylated version of PP2Ac and for the phosphomimetic mutant is T304D consistent with a role of TIPRL in the early steps of PP2A biogenesis (AU) | |
| FAPESP's process: | 16/12560-9 - Functional characterization of the role of regulatory protein TIPRL in normal and cancer cells |
| Grantee: | Ana Amélia Sanchez Iacia |
| Support Opportunities: | Scholarships in Brazil - Doctorate |