No abstract

No abstract

Retinoic acid receptor ␥ (RAR␥) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RAR␥1 and serines 66 and 68 in RAR␥2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7⅐cyclin H⅐MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RAR␥ is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RAR␥ phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RAR␥ transcriptional activity in a response gene-dependent manner.The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs) 1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily (1-4). RARs are activated by all-trans and 9-cis retinoic acid, whereas RXRs are activated by 9-cis retinoic acid only. There are three RAR (␣, ␤, and ␥) and three RXR (␣, ␤, and ␥) isotypes, and for each isotype there are at least two main isoforms that differ in their N-terminal region (1, 5, 6).As do other members of the nuclear receptor superfamily, RARs and RXRs exhibit a conserved modular structure with six variably conserved regions (A to F) (Fig. 1) (1, 5). The Nterminal A/B region of RARs contains a ligand-independent transcriptional activation function, AF-1 (7, 8). Although the B regions of the three RAR isotypes are moderately conserved, their A regions are unrelated and differ for each isoform of a given RAR isotype (5). The highly conserved C region encompasses the central DNA binding domain. The function of region F, if any, is unknown. Region E is more complex, as it contains the ligand binding domain, a dimerization interface, and the ligand-dependent transcriptional activation/repression domain AF-2 (1, 9). The activity of AF-2 is entirely dependent on the integrity of a conserved sequence referred to as the AF-2 AD core, located in ␣-helix 12 at the C-terminal end of the ligand binding domain. Ligand binding induces a major conformational change that includes helix 12 and creates a new surface for coactivator binding while corepressors are released, thus resulting in a transcriptional-competent nuclear receptor relayed to the transcriptional machinery and the chromatin template (1, 10 -12). The AF-2 and AF-1 activities synergize with each other in a response element-and promoter context-dependent manner (1,8,13).RARs and RXRs are phosphoproteins (14 -16), and their phosphorylation involves several kinases. RAR␣ can be phosphorylated in its AF-1-containing B region by the cyclin-dependent kinase cdk7 (14), which together with MAT1 and cyclin H forms the CAK complex that is found either free or as a component of the g...

The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and Drosophila tissue culture, as well as in Drosophila ovaries ex vivo, p38-activating stresses, such as H 2 O 2 and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of p38b or licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in licorne or p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and Drosophila systems in response to certain stresses and growth factors.

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.

Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RAR␥2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RAinduced phosphorylation of RAR␥2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced downregulation of the PI3K/Akt pathway targeted not only the phosphorylation of RAR␥2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.

No abstract

No abstract