In vitro kinase assay to detect STX17 phosphorylation by ULK1
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Yueguang Rong
PROVIDER: PXD039362 | iProX | Tue Jun 27 00:00:00 BST 2023
REPOSITORIES: iProX
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Project description:Mass-spectrometric analysis of an in vitro kinase assay using recombinant active ULK1 (Sigma-Aldrich, SRP5096) as kinase and GST-RIPK1(250-475) as substrate
2020-04-22 | PXD018035 | Pride
Project description:Mass-spectrometric analysis of an in vitro kinase assay using recombinant active ULK1 (Sigma-Aldrich, SRP5096) as kinase and GST-RIPK1(250-475) as substrate
2020-04-22 | PXD018078 | Pride
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
Project description:Label-free phosphoproteomics of in vitro kinase assays. Three sets of bottom-up proteomics experiments were performed.
2021-11-03 | PXD022759 | Pride
Project description:To examine whether TAF7 could be phosphorylated by DNA-PKcs directly. An in vitro kinase assays were performed. Firstly, human DNA-PKcs was purified from TGF1 treated HK2 cells by immunoprecipitation with a DNA-PKcs antibody (abcam) and IgG was used as a negative control. The purified proteins and human GST-TAF7 (purchased from proteintech) were incubated in kinase assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM MnCl2) supplemented with 200 M ATP in 50 l reactions for 30 min at 30°C. Then, phosphorylation of TAF7 was analyzed by mass spectrometry (MS)
2022-09-22 | PXD036930 |
Project description:Human papillomaviruses (HPVs) are conducive to a fraction of head and neck squamous cell carcinoma (HNSCC). Previously we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked GlcNAcylation (O-GlcNAc) transferase (OGT). Herein we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, ULK1, an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409Ser410, which is distinct from previously reported Thr635Thr754. It has been known that PKCαmediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperon-mediated autophagy (CMA) pathway. By biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at pSer423. Mutations of Ser409ASer410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperon Hsc70. Moreover, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of the TCGA database reveals that ULK1 is upregulated in HPV-positive HNSCCs and its protein level positively correlates with HNSCC patient survival. Our work demonstrates that O-GlcNAcylation of ULK1 alters to reciprocate to the environmental changes. O-GlcNAcylation of ULK1 at Ser409Ser410 stabilizes ULK1, and it might underlie the molecular mechanism of HPV-positive HNSCC patient survival.
2022-10-14 | PXD031095 | Pride
Project description:Several traumatic and neurodegenerative disorders of the CNS show axonal degeneration as a key and early pathological feature. Following a focal axonal lesion, an extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, activation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we overexpressed a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons in vitro and in the spinal cord (SC) and optic nerve (ON) in vivo. We found a significant inhibitory effect on autophagy in vitro and in vivo. While no effect on cell survival could be observed, ULK1.DN attenuated AAD after axotomy in microfluidic chambers in vitro. Models of SC injury and ON crush in vivo confirmed an attenuation of axonal degeneration by ULK1.DN. In a translational approach, we employed the selective ULK1 inhibitor SBI-0206965 (SBI) and found an attenuation of AAD after ON crush in vivo mediated by a reduction of autophagy by SBI. Quantitative proteomic profiling after ULK1.DN overexpression in vitro revealed a regulation of proteins associated with translation and splicing. A differential exon expression analysis identified the genes kinesin family member 1B (Kif1b), DNA damage inducible transcript 3 (Ddit3) and WD repeat domain 6 (Wdr6) as targets of ULK1.DN and potential mediators of its degeneration-attenuating effect. These findings reveal ULK1 as an important mediator of axonal degeneration and elucidate its function in splicing, defining it as a putative therapeutic target.
2020-09-23 | GSE123687 | GEO
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.
2014-08-26 | GSE60778 | GEO
Project description:This project involved generating three stable cell lines using a Flp-In system, each containing specific integrated constructs: "FKBPx2-myc-APEX2-ULK1", "FKBPx2-myc-APEX2-ULK1-K46I" (kinase-dead mutant), and "FKBPx2-myc-APEX2" (control). All cell lines additionally express an outer mitochondrial membrane-targeted construct consisting of truncated FIS193–152 fused to FRB (FRB-FIS193–152). Upon treatment with a heterodimerizer (rapalog), the cytosolic FKBP-tagged constructs are recruited to mitochondria via FRB-rapalog-FKBP interactions, enabling controlled mitochondrial tethering. The constructs contain APEX2 peroxidase, which catalyzes biotin-phenol biotinylation of nearby proteins upon H₂O₂ treatment, allowing proximity-dependent protein identification. The primary objective was to investigate the interactomes of wild-type ULK1 versus the kinase-dead ULK1-K46I mutant when localized to mitochondria. By comparing the biotinylated protein profiles between these conditions and the APEX2-only control, we aimed to identify ULK1-specific interacting partners and determine how kinase activity affects these interactions in the mitochondrial environment. This approach provides insights into ULK1's role in mitophagy and autophagy regulation.
2026-04-24 | PXD068381 | Pride
Project description:Finely ground leaf tissue of wild-type columbia-0 ecotype was used.
Protein extraction was performed in buffer-saturated phenol and high sucrose buffer.
Extracted proteins were incubated with purified recombinant GST or GST-BIN2 kinase.
Filter-assisted sample preparation (FASP) columns in presence of 8M urea were used to further purify proteins. Purified proteins were reduced with TCEP, alkylated with iodoacetamide and digested into peptides with trypsin and lys-C.
Digested peptides were desalted with c18 columns and labelled with 11-plex Tandem Mass Tag (TMT11) reagents.
Phosphoenrichment was performed in a two-steps tandem pipeline: High-Select TiO2 Phosphopeptide Enrichment kit (Thermo Scientific) was used first and High Select Fe-NTA Phosphoptide Enrichment kit (Thermo Scientific) was used on the flow-through obtained from first enrichment.
2020-11-17 | MSV000086462 | MassIVE