Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation.

We assign a new function to a tumor suppressor NPRL2 that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) than in cells expressing the dominant positive mutant heterodimer, RagA(GTP)/RagD(GDP). The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. Here, we propose a 'seesaw' model in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells.

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation.

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.

Genome wide expression analysis of the effect of Pinelliae Rhizoma extract on psychological stress.

Pinelliae Rhizoma has been used traditionally as an antidepressant in Oriental medicine. In this study, the effect of Pinelliae Rhizoma extract (PRe) on psychological stress was investigated in mice. The results of an elevated plus-maze experiment revealed that application of psychological stress to mice led to the development of an abnormal behavioral pattern. However, oral administration of PRe significantly reduced the abnormal behavior of mice with a recovery rate of 75.5%. To elucidate the molecular mechanism by PRe, a microarray analysis of the brains of mice was conducted. The results of this analysis revealed that 456 genes were up-regulated and 392 genes were down-regulated in response to psychological stress. The expression of most of the genes that were altered in response to psychological stress was restored to normal levels in PRe treated mice, with a recovery rate of 81.5% and 85.2% being observed for up- and down-regulated genes, respectively. Finally, when the interaction network information was analysed, the recovery rate of the core node genes (46 up- and 29 down-regulated genes) in PRe treated mice was found to be over 95%, which indicates that this final set of genes may be the effective target of PRe.

Notch signal activates hypoxia pathway through HES1-dependent SRC/signal transducers and activators of transcription 3 pathway.

We report a Notch signal-induced pathway that leads to transcriptional activation of HIF1-alpha gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-alpha and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-alpha expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.

Repression of transcriptional activity of estrogen receptor alpha by a Cullin3/SPOP ubiquitin E3 ligase complex.

The role of SPOP in the ubiquitination of ER alpha by the Cullin3-based E3 ubiquitin ligase complex was investigated. We showed that the N-terminal region of SPOP containing the MATH domain interacts with the AF-2 domain of ER alpha in cultured human embryonic 293 cells. SPOP was required for coimmunoprecipitation of ER alpha; with Cullin3. This is the first report of the essential role of SPOP in ERalpha ubiquitination by the Cullin3-based E3 ubiquitin ligase complex. We also demonstrated repression of the transactivation capability of ER alpha; in cultured mammalian cells.

Inhibition of NF-kappaB acetylation and its transcriptional activity by Daxx.

We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.

BTB/POZ domain of speckle-type POZ protein (SPOP) confers proapoptotic function in HeLa cells.

The proapoptotic function of SPOP protein was investigated in HeLa cells. HeLa cells underwent apoptosis by the overexpression of SPOP. Studies using SPOP deletion mutants suggest that BTB/POZ domain of SPOP protein is important for the induction of apoptosis in transfected cells. This study first proposes the proapoptotic aspect of the BTB/POZ domain of SPOP protein based on the finding that cells expressing the C-terminal fragment of SPOP containing the BTB/POZ domain underwent apoptosis.