Tesis doctoral de Mara Angeles Cobos Fernandez
Diacylglycerol kinases (dgk) regulate diacylglycerol-based signals by phosphorylating this key lipid intermediate to phosphatidic acid. In the present work we have analyzed the expression and regulation of dgk¿ ii in jurkat t lymphocytes and adherent cells (hek293). Rt-pcr assays shown that this isoform, which was described as a muscle specific form, is expressed in these cell lines, as well as in several mouse tissues, in the same way that dgk¿ i. However, despite of the two isoforms (dgk¿ i and ii) present the same structural domains (c1, catalytic domain, marcks domain, ankyrin repeats, pdz binding motif) dgk¿ ii doesn¿t translocate to plasma membrane in response to pkc activation in jurkat cells. Furthermore, by confocal microscopy we have observed that phosphorylation of marcks domain or c-terminal deletion, that prevent dgk¿i nuclear localization in hek293 cells, doesn¿t regulate dgk¿ ii nuclear localization. we also analyzed dgk¿ i localization in jurkat cells conjugated with antigen-presenting cells (apcs) by confocal microscopy and we observed that dgk¿ i marcks domain phosphorylation is necessary for dgk¿ i translocation to plasma membrane. The dgk¿ i c-terminal region (ankyrin repeats and pdzbm) blocks the enzyme plasma membrane translocation in jurkat-apc conjugated cells and dgk¿ i marcks phosphorylation allows to eliminate this restriction. the relationship between cell-cycle progression and dgk¿ i nuclear localization was also studied by confocal microscopy in this work. Dgk¿ i is accumulated in the nuclei of hek293 quiescent cells whiles the enzyme localyzes to the cytosol when cells entry to cycle in respond to serum addition and at the end of mytosis (citokinesis). We also examined the regulation of dgk¿ i nuclear localization in this cells analyzing subcellular localization of several mutants by confocal microscopy. We found that phosphorylation of dgk¿ i marcks domain prevent the protein nuclear import even in quiescent cells, whiles the dgk¿ i c1 domains are necessary to cytosolic protein localization in respond to serum addition. However the dgk¿ i nuclear localization seems to be independent of its catalytic activity or pdzbm. the treatment of hek293 cells with the nuclear export inhibitor leptomycin b shown that dgk¿ i nuclear export is mediated by the exportin crm-1. Analyzing the localization of dgk¿ i c-terminal deleted mutant (dgk¿ i¿ank) in this conditions we have observed that this region regulates dgk¿ i nuclear accumulation because it blocks crm-1-mediated dgk¿ i nuclear export. finally the function of nuclear dgk¿ i was analyzed in nih3t3 cells that were transfected in a stable manner. We analyzed the cell cycle progression and the protein expression of some cell-cycle regulators in nih3t3 cells over-expressing dgk¿ i versus nih3t3 cells over-expressing dgk¿ i¿ank mutant that doesn¿t accumulate in cell nucleus. By western blot we observed that dgk¿ i nuclear over-expression down-regulates the levels of p21 and cyclin d1 proteins and its increase in respond to serum. P21 and cyclin d1 are pkc nuclear effectors and its mrna-hur binding in the nucleus is required for mrna stabilization and protein expression which leads the increase of p21 and cyclin d1 levels. This process can be regulated by nuclear pkc and we have observed, by hur ip-rt-pcr assays, that over-expression of nuclear dgk¿ i inhibit hur binding of p21 and cyclin d1 mrnas. So in this work we have found a new role for nuclear dgk¿ i as a negative regulator of hur mediated post-transcriptional regulation of proteins such as p21 and cyclin d1. iii.
Datos académicos de la tesis doctoral «Estudio de la regulacion de la localizacion subcelular de diacilglicerol quinasa y su funcion en nucleo«
- Título de la tesis: Estudio de la regulacion de la localizacion subcelular de diacilglicerol quinasa y su funcion en nucleo
- Autor: Mara Angeles Cobos Fernandez
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 02/07/2010
Dirección y tribunal
- Director de la tesis
- Isabel Merida San Roman
- Tribunal
- Presidente del tribunal: manuel Ortíz de landázuri busca
- José Alberto García sanz (vocal)
- Mª luz Martinez chantar (vocal)
- María senena Corbalan garcia (vocal)