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ULK1 regulated phosphosites in mouse and human cells

ABSTRACT: (1)In vivo SILAC analyses of mouse embryonic fibroblasts (MEFs, triple labeling) were performed comparing phosphorylation events. ULK1 double knock out MEFs (VC) were compared to double knock out MEFs expressing human ULK1 (RE) in fed (DMEM) and starvation (HBSS) conditions. (2) In vivo SILAC analyses of A549 cells (triple labeling) were performed comparing phosphorylation events (195 raw files labeled ""A549""). A549 cells in fed conditions (DMEM) were compared to starved cells (HBSS) and cells treated with rapamycin (Rapa).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Fibroblast

SUBMITTER: Joern Dengjel

LAB HEAD: Joern Dengjel

PROVIDER: PXD022303 | Pride | 2021-11-03

REPOSITORIES: Pride

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ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy.

Hu Zehan Z Sankar Devanarayanan Siva DS Vu Bich B Leytens Alexandre A Vionnet Christine C Wu Wenxian W Stumpe Michael M Martínez-Martínez Esther E Stork Björn B Dengjel Jörn J

Cell reports 20210901 13

The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation ...[more]

PMID: 34592149

Similar Datasets

ULK1 regulated phosphatases in mouse and human cells

Project description:In the project “ULK1 regulated phosphatases” by Zehan Hu, Devanarayanan Siva Sankar, Björn Stork and Jörn Dengjel two sets of bottom-up MS-based chemical proteomics experiments were performed in which phosphatase complexes were enriched based on their activity using microcystin-LR coupled sepharose beads. (1) Three biological replicates of MEFs (triple labeling) were performed comparing phosphatase activity (36 raw files labeled "2018"). ULK1 MEFs were compared DKO MEFs, both in rapamycin conditions. Empty beads were used as negative control (Ctrl). Following experiments were performed: (2) Three biological replicates of A549 cells (triple labeling) were performed comparing phosphatase activity (30 raw files labeled "A549"). A549 cells in fed conditions (DMEM) were compared to rapamycin treated cells (Rapa). Empty beads were used as negative control (Ctrl).

2021-11-03 | PXD022383 | Pride

Central Role of ULK1 in Type I Interferon Signaling

Project description:We provide evidence that the Unc-51-like kinase 1 (ULK1) is phosphorylated and activated during engagement of the Type I IFN receptor. Our studies demonstrate that the function of ULK1 controls expression of key interferon stimulated genes (ISGs) that mediate important biological functions, including anti-viral and antineoplastic responses. Total RNA was isolated from untreated and IFNβ-treated Ulk1/2+/+ and Ulk1/2-/- mouse embryonic fibroblasts (MEFs). Cells were treated for 6 hours with or without mouse IFNβ (2500 IU/ml).

2014-08-26 | E-GEOD-60778 | biostudies-arrayexpress

Mus musculus

Project description:DMEM treated WT and PKBa -/- MEFs

| PRJNA92217 | ENA

Autophagy and alternative NF-kappaB inhibition by RNAi against RELB, ATG5 or ULK1 in A549 human cancer cells

Project description:In order to understand the contribution of autophagy and alternative NF-kappaB signalling to transcriptional output in A549 lung cancer cells, RNAi was used to knockdown the expression of core autophagy genes (ATG5, ULK1) or the key subunit of alternative NF-kappaB RELB. Seven samples were prepared for each biological replicate; two sequence independent non-targeting control siRNAs were transfected as a negative control. Three sequence independent siRNAs targeting the core autophagy machinery were transfected (ATG5 si, ATG5 si(2), ULK1 si). N.B. ATG5 si(2) only partially ablates Atg5 protein expression whereas ATG si has greater efficacy. Two sequence independent siRNAs targeting RELB were transfected (RELB si, RELB si(2) ). Three independent biological replicates of all conditions were obtained, by performance of the experiment on different days.

2015-09-18 | E-GEOD-73158 | biostudies-arrayexpress

Understanding the physiological role of asparaginyl endopeptidase (AEP)/Legumain

Project description:Understanding the regulation of lysosomal proteolytic/hidrolytic capacity has been recently advanced in a huge manner by the discovery of the lysosome-mTOR-TFEB pathway connecting nutrient sensing, autophagy and de novo lysosome generation. But, how lysosomes can adjust their proteolytic/hidrolytic capacity to meet varying conditions (more or less substrate) under normal fed conditions is not known. We have used AEP as a proxy to understand how lysosomes can compensate the lack of a key lysosomal protease to meet the requirements under physiological, fed conditions. To do this, we did compare cytoplasmic, membrane and nuclear fractions of WT and AEP MEFs that were expanded in SILAC media. AEP knock-out mice show pale kidney, with abnormal proliferation of the epithelial proximal tubular cells. In an attempt to understand the role of AEP in the kidney, we did compare kidney obtained from WT and AEP mice using kidneys from WT mice that were fed with SILAC food. Also, in an attempt to reproduce the changes observed in the absence of AEP in kidney and MEFs, we treated WT MEFs grown in SILAC media with MVO26630, specific inhibitor of AEP, for 12 and 48 hours.

2019-01-09 | PXD011473 | Pride

DNA methylation analysis of mouse embryonic fibroblasts (MEFs) and Thymidine treatment-induced MEFs (induced cells).

Project description:Investigation of DNA methlation changing in thymidine treatment-induced MEFs. Mouse embryonic fibroblasts (MEFs) were obtained at E14.5 embryos and supplied with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 15% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Potential hematopoietic progenitor cells (CD45+CD41+CD31+c-Kit+) were negatively selected with Biotin labeled anti-CD45, anti-CD31, anti-CD41 and anti-c-Kit antibodies by magnetic beads. Hereafter, treated MEFs are referred to as 4neg MEFs for initiation of induction. 4neg MEFs were seeded on mitomycin (MMC)-treated MEFs as feeder cells and cultured in DMEM with 15% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 μM thymidine as induction medium for 2 days followed with analysis

2020-01-28 | GSE144272 | GEO

Transcription profiling of differentiating mouse embryonic fibroblasts generated from wild type and PKBa-/- mice to study the role of PKBa in adipose differentiation.

Project description:Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos obtained from heterozygous PKBa mice intercrosses (Yang et al., 2003). Briefly, after dissection of head and visceral organs for genotyping, embryos were minced and trypsinized for 30 min at 37°C. Embryonic fibroblasts were then plated and maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) (Life Technologies), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with wild-type and PKBa-/- MEFs between 15-20 passages. To induce adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with DMEM supplemented with 10% FCS, 8 mg/ml biotin, 4 mg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone and 10 mg/ml insulin (all from Sigma). Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer's instructions. Experiment Overall Design: Time course of DMEM treated WT and PKBa -/- MEFs in quadruplicate

2007-12-01 | E-GEOD-2746 | biostudies-arrayexpress

Autophagy and alternative NF-kappaB inhibition by RNAi against RELB, ATG5 or ULK1 in A549 human cancer cells

2015-09-18 | GSE73158 | GEO

Expression analysis of mouse embryonic fibroblasts (MEFs) and Thymidine treatment-induced MEFs (induced cells)

Project description:Investigation of differentially expressed genes in thymidine treatment-induced MEFs. Mouse embryonic fibroblasts (MEFs) were obtained at E14.5 embryos and supplied with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 15% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Potential hematopoietic progenitor cells (CD45+CD41+CD31+c-Kit+) were negatively selected with Biotin labeled anti-CD45, anti-CD31, anti-CD41 and anti-c-Kit antibodies by magnetic beads. Hereafter, treated MEFs are referred to as 4neg MEFs for initiation of induction. 4neg MEFs were seeded on mitomycin (MMC)-treated MEFs as feeder cells and cultured in DMEM with 15% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 μM thymidine as induction medium for 6 days. Cells were then trypsinized and re-seeded to new feeder cells for another 6 days. Cells were positively selected with Biotin labeled anti-CD45 by magnetic beads followed with analysis.

2020-01-11 | GSE143418 | GEO

Effect of endophilin A deficiency in mouse hippocampus

Project description:We tested how complete or partial loss of endophilin A1, A2 and A3 affects gene expression in mouse hippocampus. Total loss of endophilin (triple knock-outs, TKO) was assessed in newborn mice, since the TKO mice only survive only several hours after birth. Partial loss of endophilin (endoA1,A2 double knock-out, DKO) was assessed between

2016-08-17 | GSE85702 | GEO

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