Dataset Information

MCF-7 phosphoproteome LC-MS/MS

ABSTRACT: MCF-7 cells were stimulated with TNF-alpha in order to identify IKKb substrates. conditions: TNF alpha stimulation TNF alpha stimulation + SC-514 IKK (kinase dead mutant) + TNF alpha stimulation IKK(WT) + TNF alpha stimulation basal Already validated IKK substrates were used to train random forest and to predict new substrates. Among other interesting candidates we validated AEG-1 (S298) as an IKKb substrate. We provide evidence that IKKb-mediated AEG-1 phosphorylation is essential for IkBa degradation as well as NF-kB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. (replicate 1 out of at least 2)

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Hendrik Nolte

LAB HEAD: Marcus Krüger

PROVIDER: PXD001873 | Pride | 2018-10-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20140313_HN_Hendrik_Ramesh_PhosphoExp1_L_basal_M_Tnf_H_TnfPlusInh_01.raw	Raw
	20140313_HN_Hendrik_Ramesh_PhosphoExp1_L_basal_M_Tnf_H_TnfPlusInh_02.raw	Raw
	20140313_HN_Hendrik_Ramesh_PhosphoExp1_L_basal_M_Tnf_H_TnfPlusInh_03.raw	Raw
	20140313_HN_Hendrik_Ramesh_PhosphoExp1_L_basal_M_Tnf_H_TnfPlusInh_04.raw	Raw
	20140313_HN_Hendrik_Ramesh_PhosphoExp1_L_basal_M_Tnf_H_TnfPlusInh_05.raw	Raw

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Publications

Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate.

Krishnan Ramesh K RK Nolte Hendrik H Sun Tianliang T Kaur Harmandeep H Sreenivasan Krishnamoorthy K Looso Mario M Offermanns Stefan S Krüger Marcus M Swiercz Jakub M JM

Nature communications 20150407

The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteom ...[more]

PMID: 25849741

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Project description:Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas. It is characterized by the presence of rare Hodgkin and Reed/Sternberg (HRS) cells embedded in an extensive inflammatory infiltrate. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in HRS cells which transcriptionally regulates expression of multiple anti-apoptotic factors and pro-inflammatory cytokines plays a central role in the pathogenesis of cHL (1, 2). In non-stimulated condition, NF-kappaB proteins are rendered inactive by binding to their inhibitors (IkappaB s), which sequester them in the cytoplasm. Stimulation of multiple receptors activates the IkappaB kinase (IKK) complex that phosphorylates IkappaB at two specific serine residues, followed by its ubiquitination and proteasomal degradation, thereby releasing NF-kappaB proteins and allowing their nuclear translocation (3). Recently, two studies provided further insights into the molecular mechanisms of IKK activation upon TNF stimulation (4, 5). Activation of the IKK complex and subsequent NF-kappaB activation requires Lys63 polyubiquitination of RIP1, a kinase which is recruited to the receptor upon TNF stimulation. IKK-ï§ï (NEMO), the regulatory subunit of the IKK complex, specifically recognizes these Lys63-linked polyubiquitins attached to RIP1 and thereby activates IKK and NF-kappaB (4, 5). A20 is an ubiquitin-modifying enzyme that inhibits NF-kappaB activation in succession of tumor necrosis factor (TNF) receptor and Toll-like receptor induced signals (6-8). This enzyme removes Lys63 linked ubiquitin chains from RIP1 and adds Lys48 polyubiquitins to RIP1, thereby targeting this factor for proteasomal degradation, thus explaining the molecular mechanism of NF-kappaB inhibition by A20 (6). A20 likely inhibits NF-kappaB acitivity also by additional means, including interaction with TRAF1 and TRAF2 (9). SNP 6.0 array (Affymetrix) analyses were performed according to the manufacturer's directions on DNA extracted from three Hodgkin cell lines (L1236, HDLM-2, U-HO1), HapMap samples included in the Genotyping Console Software 3.0 were used as references.