Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.

LDL receptor related proteins 5 and 6 (LRP5/6) and their Drosophila homolog Arrow are single-span transmembrane proteins essential for Wnt/beta-catenin signaling, likely via acting as Wnt coreceptors. How Wnt activates LRP5/6/Arrow to initiate signal transduction is not well defined. Here we show that a PPPSP motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, is necessary and sufficient to trigger Wnt/beta-catenin signaling. A single PPPSP motif, upon transfer to the LDL receptor, fully activates the Wnt pathway, inducing complete axis duplication in Xenopus and TCF/beta-catenin-responsive transcription in human cells. We further show that Wnt signal-ing stimulates, and requires, phosphorylation of the PPPSP motif, which creates an inducible docking site for Axin, a scaffolding protein controlling beta-catenin stability. Our study identifies a critical signaling module and a key phosphorylation-dependent activation step of the Wnt receptor complex and reveals a unifying logic for transmembrane signaling by Wnts, growth factors, and cytokines.

Canonical Wnt/␤-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/␤-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/␤-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate ␤-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/␤-catenin signaling.

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.

No abstract

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by ϳ4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3␤ (GSK-3␤), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3␤ at Ser-9, which negatively regulates the kinase activity, GSK-3␤ exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.The physiology and behavior of living organisms from bacteria to humans show daily fluctuations, and those controlled by autonomous clocks are termed circadian rhythms (1, 2). These rhythms are synchronized with (entrained to) environmental time cues such as light, and the rhythms are sustained with a period of ϳ24 h even in the absence of the time cues. In mammals, the suprachiasmatic nucleus in the anterior hypothalamus serves as the central clock of the circadian timing system (3-5). Peripheral tissues throughout the body also have circadian clocks, and both the central and peripheral clocks generate the 24-h rhythm with molecular machinery very similar to each other (6 -8).The molecular mechanism of the circadian oscillator has been investigated extensively by genetic and molecular studies on Drosophila and mice. In the mouse molecular clock, a heterodimer of the two transcription factors, CLOCK and BMAL1, activates E-box-dependent transcription of two cryptochrome genes, mCry1 1 and mCry2, and three period genes, mPer1, mPer2, and mPer3 (9, 10). Translated mCRY and mPER proteins translocate to the nucleus where mCRY proteins act as predominant negative regulators by interacting directly with CLOCK/BMAL1 heterodimer to inhibit the transactivation from the E-box (10, 11). The negative regulation in turn results in decrease of the protein levels of mCRYs and mPERs and allows the molecular cycle to start again with the activation of the E-box-dependent transcription. Importantly, mice lacking both mCry1 and mCry2 exhibit arrhythmic behavior immediately after being placed in constant darkness (12), indicating their c...

Epitaxial (111) films of Cu, Ni, and CuxNiy on α−Al2O3 (0001) for graphene growth by chemical vapor deposition J. Appl. Phys. 112, 064317 (2012) Studies on atomic layer deposition Al2O3/In0.53Ga0.47As interface formation mechanism based on air-gap capacitance-voltage method Appl. Phys. Lett. 101, 122102 (2012) Temperature dependence of chemical-vapor deposition of pure boron layers from diborane Appl. Phys. Lett. 101, 111906 (2012) Growth of continuous and ultrathin platinum films on tungsten adhesion layers using atomic layer deposition techniques Appl. Phys. Lett. 101, 111601 (2012) Black silicon with controllable macropore array for enhanced photoelectrochemical performance

The population dynamics of plants in a lattice structured habitat are studied theoretically. Plants are assumed to propagate both by producing seeds that scatter over the population and by vegetative reproduction by extending runners, rhizomes, or roots, to neighboring vacant sites. In addtion, the seed production rate may be dependent on the local density in the neighborhood, indicating beneficial or harmful crowding effects. Two sets of population dynamical equation(s) are derived: one based on mean‐field approximation and the other based on pair approximation (tracing both global and local densities simultaneously). We examine the accuracy of these approximate dynamics by comparing them with direct computer simulation of the stochastic lattice model. Pair approximation is much more accurate than mean‐field approximation. Mean‐field approximation overestimates the parameter range for persistence if crowding effects on seed production are harmful or weakly beneficial, but underestimates it if crowding effects are highly beneficial. Dynamics may show bistability (both population persistence and extinction) if the effect of crowding is strongly beneficial. If there is a linear trade‐off between seed production and vegetative reproduction, the equilibrium abundance of the population may be maximised by a mixture of seed production and vegetative reproduction, rather than by pure seed production or by pure vegetative reproduction. This result is correctly predicted by pair approximation but not by mean‐field approximation.