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

Intervention to increase physical activity and healthy eating among under-represented adolescents: GOAL trial protocol

Introduction To reduce obesity-related disparities, reaching economically disadvantaged and/or minority status adolescents to assist them in meeting physical activity (PA) and nutrition recommendations is important. To address the problem, a 16-week intervention called Guys/Girls Opt for Activities for Life (GOAL) was designed. The purpose of this randomised controlled trial is to evaluate any effect of the intervention, compared with a control condition, on improving: (1) adolescents’ % body fat (primary outcome), moderate-to-vigorous PA (MVPA), diet quality and cardiorespiratory fitness from 0 to 4 months; (2) body mass index (BMI), overweight/obesity percentage and quality of life from 0 to 4 months and to 13 months; and (3) perceived social support, self-efficacy and motivation from 0 to 4 months with evaluation of any mediating effect on adolescent PA and diet quality. An exploratory aim is to evaluate any effect of the intervention, compared with the control, on improving parents’/guardians’ home environment, MVPA and diet quality from 0 to 4 months; and BMI from 0 to 4 months and to 13 months.Methods and analysis Adolescents (fifth to eighth grade) in 14 schools located in underserved urban communities are randomly assigned to the intervention or usual school offerings. One parent per adolescent is enrolled (882 dyads total). Cohort 1 includes four schools (2022–2023). Cohorts 2 and 3 include 5 schools in 2023–2024 and 2024–2025, respectively. The 16-week intervention has three components: (1) after-school GOAL club for adolescents to engage in PA and healthy eating/cooking activities; (2) three parent–adolescent meetings to empower parents to assist adolescents; and (3) GOAL social networking website for parents to share how they helped their adolescent.Ethics and dissemination The Michigan State University Biomedical Institutional Review Board provided ethical approval for the study. Findings will be shared via the trial registration database, peer-reviewed publications, conferences and community-oriented strategies.Trial registration number NCT04213014

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

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

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