C-RAF activation promotes BAD poly-ubiquitylation and turn-over by the proteasome

doi:10.1016/j.bbrc.2008.03.141

Biochemical and Biophysical Research Communications

Volume 370, Issue 4, 13 June 2008, Pages 552-556

https://doi.org/10.1016/j.bbrc.2008.03.141 Get rights and content

Abstract

BAD, a member of the BCL2 family, exhibits an original mode of regulation by phosphorylation. In the present report, we examine the role of the kinase C-RAF in this process. We show that the inducible activation of C-RAF promotes the rapid phosphorylation of BAD on Serine-112 (Ser-75 in the human protein), through a cascade involving the kinases MEK and RSK. Our findings reveal a new aspect of the regulation of BAD protein and its control by the RAF pathway: we find that C-RAF activation promotes BAD poly-ubiquitylation in a phosphorylation-dependent fashion, and increases the turn-over of this protein through proteasomal degradation.

Section snippets

Materials and methods

Cell culture and reagents. All cell lines were cultivated in DMEM supplemented with 10% FCS, 2 mM l-glutamine, and antibiotics (penicillin–streptomycin). The NIH 3T3 C-RAF BXB-ER cell line is a clonal derivative of the NIH 3T3 fibroblast that expresses the fusion protein C-RAF BXB-ER at stable, low level [13]. In this cell line, the estrogen agonist 4-hydroxytamoxifene (4-OHT) stimulates C-RAF kinase activity.

MG132 came from Sigma, U0126 from Promega, BAY43-9006 from Bayer. BI-D1870 was a gift

C-RAF stimulation promotes the rapid phosphorylation of BAD

We used an inducible system, the cell line NIH 3T3 C-RAF-BXB-ER to study the effects of the C-RAF kinase pathway on BAD (Fig. 1). We noticed that C-RAF activation promotes the rapid phosphorylation of BAD on a single residue, Ser-112 (Fig. 1A and B). 4-OHT did not induce BAD phosphorylation in NIH 3T3 cells expressing a control vector or a kinase-dead version of C-RAF BXB-ER (Fig. 1C). In agreement with the dependency of BAD phosphorylation on RAF catalytic activity, BAD phosphorylation on

Acknowledgments

We thank the people who contributed reagents used here. We thank in particular Ulf R. Rapp for initiating the study of BAD phosphorylation, providing key reagents and support. This work was funded through J.F.’s thesis allocation (Graduate College GRK1141/1, DeutscheForschung Gemeinschaft, and French-German University ED-31-04).

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C-RAF activation promotes BAD poly-ubiquitylation and turn-over by the proteasome

Abstract

Section snippets

Materials and methods

C-RAF stimulation promotes the rapid phosphorylation of BAD

Acknowledgments

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Cell

Cell

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Curr. Biol.

J. Biol. Chem.

Cancer Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.