To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

In canonical Wnt signaling, Dishevelled (Dvl) is a critical cytoplasmic regulator that releases β-catenin from degradation. Here, we find that Dvl and c-Jun form a complex with β-catenin–T-cell factor 4 (TCF-4) on the promoter of Wnt target genes and regulate gene transcription. The complex forms via two interactions of nuclear Dvl with c-Jun and β-catenin, respectively, both of which bind to TCF. Disrupting the interaction of Dvl with either c-Jun or β-catenin suppresses canonical Wnt signaling–stimulated transcription, and the reduction of Dvl diminished β-catenin–TCF-4 association on Wnt target gene promoters in vivo. Expression of a TCF-Dvl fusion protein largely rescued the c-Jun knockdown Wnt signaling deficiency in mammalian cells and zebrafish. Thus, we confirm that c-Jun functions in canonical Wnt signaling and show that c-Jun functions as a scaffold in the β-catenin–TCFs transcription complex bridging Dvl to TCF. Our results reveal a mechanism by which nuclear Dvl cooperates with c-Jun to regulate gene transcription stimulated by the canonical Wnt signaling pathway.

mTORC2 (mammalian target of rapamycin complex 2) plays important roles in signal transduction by regulating an array of downstream effectors, including protein kinase AKT. However, its regulation by upstream regulators remains poorly characterized. Although phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) is known to regulate the phosphorylation of AKT Ser 473 , the hydrophobic motif (HM) site, by mTORC2, it is not clear whether PtdIns(3,4,5)P 3 can directly regulate mTORC2 kinase activity. Here, we used two membrane-docked AKT mutant proteins, one with and the other without the pleckstrin homology (PH) domain, as substrates for mTORC2 to dissect the roles of PtdIns(3,4,5)P 3 in AKT HM phosphorylation in cultured cells and in vitro kinase assays. In HEK293T cells, insulin and constitutively active mutants of small GTPase H-Ras and PI3K could induce HM phosphorylation of both AKT mutants, which was blocked by the PI3K inhibitor LY294002. Importantly, PtdIns(3,4,5)P 3 was able to stimulate the phosphorylation of both AKT mutants by immunoprecipitated mTOR2 complexes in an in vitro kinase assay. In both in vivo and in vitro assays, the AKT mutant containing the PH domain appeared to be a better substrate than the one without the PH domain. Therefore, these results suggest that PtdIns(3,4,5)P 3 can regulate HM phosphorylation by mTORC2 via multiple mechanisms. One of the mechanisms is to directly stimulate the kinase activity of mTORC2. mTOR (mammalian target of rapamycin) is an evolutionarily conserved Ser/Thr protein kinase, which plays an integral role in coordinating cell growth and division in response to growth factors, nutrients, and other microenvironmental changes of the cell (1, 2). This kinase is found in mTORC1 and mTORC2, two structurally and functionally distinct complexes, which are also conserved from yeasts to mammals. In addition to shared mTOR and mLST8, distinct components of these two complexes have been reported: Raptor and PRAS40 for mTORC1 (3-7) and Rictor, SIN1, and PRR5/PRR5L for mTORC2 (8 -12). Two mTOR complexes have distinct cellular functions and are regulated differently. The mTORC1 activity is sensitive to inhibition by rapamycin, which regulates diverse cellular processes, including protein synthesis, ribosome biogenesis, transcription, and autophagy, some of which are regulated through its direct substrates S6 kinases and the eukaryotic initiation factor 4E-binding protein 1 (13-16).The mTORC2 activity is resistant to rapamycin, at least in short term treatment (17,18). It phosphorylates the hydrophobic motif (HM) 2 sites of several AGC kinases, including AKT, PKC, and SGK1, to activate their kinase activities (19 -23). Studies using cells derived from Rictor, mSin1, or mLST8 knock-out mice show that intact mTORC2 is necessary for AKT HM phosphorylation (12, 24 -26). In addition, mTORC2 phosphorylates the turn motif site of AKT and regulates AKT stability, but this phosphorylation is independent of growth factor regulation (19,21). Moreover, mTORC2 can also regulate acti...

Csi1 promotes centromere clustering by linking centromeres to the SUN domain protein Sad1 in the nuclear envelope.

No abstract

All living organisms are constantly exposed to stresses from internal biological processes and surrounding environments, which induce many adaptive changes in cellular physiology and gene expression programs. Unexpectedly, constitutive heterochromatin, which is generally associated with the stable maintenance of gene silencing, is also dynamically regulated in response to stimuli. In this review we will discuss the mechanism of constitutive heterochromatin assembly, its dynamic nature, and its responses to environmental changes.

Heterochromatin, a highly compact chromatin state characterized by histone H3K9 methylation and HP1 protein binding, silences the underlying DNA and influences the expression of neighboring genes. However, the mechanisms that regulate heterochromatin spreading are not well understood. In this study, we show that the conserved Mst2 histone acetyltransferase complex in fission yeast regulates histone turnover at heterochromatin regions to control heterochromatin spreading and prevents ectopic heterochromatin assembly. The combined loss of Mst2 and the JmjC domain protein Epe1 results in uncontrolled heterochromatin spreading and massive ectopic heterochromatin, leading to severe growth defects due to the inactivation of essential genes. Interestingly, these cells quickly recover by accumulating heterochromatin at genes essential for heterochromatin assembly, leading to their reduced expression to restrain heterochromatin spreading. Our studies discover redundant pathways that control heterochromatin spreading and prevent ectopic heterochromatin assembly and reveal a fast epigenetic adaptation response to changes in heterochromatin landscape.DOI: http://dx.doi.org/10.7554/eLife.06179.001

Mammalian target of rapamycin complex (MTORC) 2 phosphorylates AGC protein kinases including PKC and regulates cellular functions including cell migration. However, its regulation remains poorly understood. Here we show that LPA induces two phases of PKCδ hydrophobic motif (HM) phosphorylation. The late phase is mediated by Gα12, which specifically activates ARAF, leading to upregulation of the expression of an E3 ubiquitin ligase RFFL and subsequent ubiquitination and degradation of PRR5L. Destabilization of PRR5L, a suppressor of mTORC2-mediated HM phosphorylation of PKCδ, but not AKT, results in PKCδ HM phosphorylation and activation. This Gα12-mediated pathway is critically important for fibroblast migration and pulmonary fibrosis development. Thus, our study unravels a signaling pathway for mTORC2 regulation and fibroblast migration.