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

Abstract

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 phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.

Astrocyte elevated gene-1 in relation to colorectal cancer development and radiotherapy response

“…Later, treatment with TNF-α showed to induces AEG-1 phosphorylation at Ser298 by the kinase IKKβ (210). Furthermore, TNF-α treatment or AEG-1 up-regulation increased the interaction with the NF-κB p65 subunit and nuclear translocation (198,204,205,210). We could confirmed the increased AEG-1 expression and translocation to the nucleolus upon TNF-α treatment in the SW480 cell line (unpublished data).…”

“…Class I proteins have the NH2-terminus exposed to the exterior site and the COOHterminal exposed to the cytoplasmic site, while it is the other way around for class II proteins (109,199 (109,(204)(205)(206)(207)(208). Furthermore, several post-translational modification sites were predicted, including phosphorylation, ubiquitination, and acetylation, while only the phosphorylation of AEG-1 at Ser298 was experimentally validated and showed biological significance (209,210).…”

IKKβ promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3

“…More recently, a high-throughput screen combining quantitative MS with random forest bioinformatics to dissect the TNFα-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells revealed dozens of potential IKKβ substrates (Krishnan et al 2015). Of significance, PFKFB3 phosphorylation was identified in this study, providing independent confirmation that PFKFB3 is a putative IKKβ substrate.…”

Astrocyte elevated gene-1 (AEG-1) and the A(E)Ging HIV/AIDS-HAND

“…Similarly, AEG-1 knockdown in astrocytes exposed to IL-1β and TNF-α reduced NF-κB activation and signaling, suggesting that AEG-1 can be utilized as a target associated with astrocyte-mediated neuroinflammation (Vartak-Sharma et al , 2014). This observation is further supported by a recent phosphoproteomics study conducted in breast cancer cells, wherein it was demonstrated that AEG-1 is a direct target of IKKβ at serine 298 and that AEG-1 phosphorylation is essential for IκBα degradation, as well as, NF-κB-dependent gene expression (Krishnan et al , 2015). …”