Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6

BACKGROUND: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway. METHODOLOGY/PRINCIPAL FINDINGS: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. CONCLUSIONS: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway

Casein Kinase 1 Proteomics Reveal Prohibitin 2 Function in Molecular Clock

Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2

Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation

Time-dependent CK1δ and CK1ε proteomics.

<p>(<b>A</b>) Illustration of screen. Cells stably expressing dually-tagged (yellow hexagon) CK1 were treated with dexamethasone (DEX) and harvested at different time points. Protein complexes were stabilized with DSP prior to lysis and purification for peptide identification by LC-MS/MS. (<b>B</b>) HA immunoblot showing DSP efficacy and the effect of dexamethasone on CK1ε complexes. (<b>C</b>) Diagram showing proteins pulled out at the different time points by CK1δ and CK1ε. Numbers represent hours after DEX that cells were harvested. Open circles were pulled out with both CK1δ and CK1ε (orange circles are known circadian proteins). Closed circles were pulled out with CK1δ alone (black-fill) or CK1ε alone (red-fill).</p

Cell-based kinase and Lumicycle assays.

<p>(<b>A</b>) Autoradiography (P³²) and immunoblots of HA-tagged proteins (green) and GFP-tagged CK1δ or CK1ε (red). Graph <i>inset</i> showing percent change of SAPS3 phosphorylation by CK1δ and CK1ε (n = 2). (<b>B</b>) Results from LumiCycle assays in cells transfected with indicated siRNAs. (<b>C</b>) Representative traces after cell synchronization of control (gray), <i>CRY2</i> (green) and <i>PHB2</i> (white) siRNAs (<b><i>left</i></b>). Graph showing effects of control siRNA, <i>CRY2</i> siRNA, two <i>PHB2</i> siRNAs (<i>PHB</i>2.1 & <i>PHB</i>2.2) and three <i>PHB1</i> siRNAs (<i>PHB</i>1.1, <i>PHB</i>1.2, <i>PHB</i>1.3) (<i>right</i>). (Sample size indicated by number inside each bar, ** indicates p<0.001 following regression analysis. Error bars indicate SEM).</p

PHB2 is molecular clock component.

<p>(<b>A</b>) Representative graph (n = 3) of M34-luciferase reporter assays following transient cotransfections of M34-luc, renilla, GFP and indicated siRNAs without (left) or with (right) <i>BMAL1</i> and <i>CLOCK</i>. (<b>B</b>) Representative graph (n = 3) showing transcript levels of <i>PER2</i> and <i>PHB2</i> 24 h after transfecting cells with control, <i>PHB2</i>, <i>CK1δ</i>, <i>CK1ε</i> and <i>PER2</i> siRNA. (<b>C</b>) Graph (n = 3) showing the effect of indicated siRNAs without (left, marked as control) or with (right, marked as <i>PHB2</i>) <i>PHB2</i> siRNA on PHB2 protein levels relative to ACTIN. (<b>D</b>) Illustration summarizing molecular mechanism based on our results where shapes indicate proteins. (* indicates p<0.05; ** indicates p<0.01; determined by student t-test analysis. Error bars indicate STDEV).</p