The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

Ikram El Yazidi-Belkoura,Gabriel Bidaux,Christian Slomianny,Franck Vandermoere,Hubert Hondermarck,Jérôme Lemoine,Eric Adriaenssens,Yohann Demont

doi:10.1074/jbc.m510003200

Ikram El Yazidi-Belkoura, Gabriel Bidaux + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m510003200

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Abstract

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappaB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

Highlights

The valosin-containing protein (VCP)3 belongs to the AAA (ATPases associated with various cellular activities) family, the members of which are characterized by having highly conserved ATPase domain(s) with high sequence similarities and ring structures consisting of homo-oligomers [1, 2]
The serine/threonine kinase Akt is increasingly appearing as a general mediator of cell survival signaling, with high potentialities as a drug target in various diseases characterized by a deregulation of the balance between cell apoptosis and cell growth, such as cancer [10]
Akt activation of I␬B kinase results in the phosphorylation and subsequent degradation of I␬B through the ubiquitin-mediated proteasome proteolysis, allowing nuclear factor ␬B (NF-␬B) to translocate into the nucleus, where it stimulates target gene expression [11]

Summary

EXPERIMENTAL PROCEDURES

Materials—Cell culture reagents were purchased from BioWhittaker. Recombinant human fibroblast growth factor-2 (FGF-2) was from R&D systems. Apoptosis of MCF-7 cells was induced by treatment with ceramide analogue C2 (10 ␮M for 24 h), previously described as a pro-apoptotic agent for human breast cancer cells [4]. The determinations of both the NF-␬B activation (luciferase reporter assay) and the proportion of cells in apoptosis (Hoechst staining) were performed as described [3]. For apoptosis induction by chemotherapeutic agents, MCF-7 breast cancer cells were treated for 3 h with 5-fluorouracil (100 ␮M), camptothecin (0.1 nM), or etoposide (10 ␮M) and replaced in new culture medium. To inhibit PI3K/Akt, cells were pretreated for 3 h by 100 nM wortmannin before FGF-2 stimulation.

VCP Is a Target of Akt Signaling

Peptide sequence consistent with mass

RESULTS

DISCUSSION