Grb10 and Active Raf-1 Kinase Promote Bad-dependent Cell Survival

• Published in: The Journal of biological chemistry Vol. 282; no. 30; pp. 21873 - 21883
• Main Authors: Kebache, Sem, Ash, Josée, Annis, Matthew G., Hagan, John, Huber, Maria, Hassard, Jennifer, Stewart, Colin L., Whiteway, Malcolm, Nantel, André
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.07.2007; American Society for Biochemistry and Molecular Biology
• Subjects: Adenocarcinoma; Animals; Apoptosis; bcl-Associated Death Protein - physiology; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Survival; Epithelial Cells - cytology; Epithelial Cells - physiology; Female; Fibroblasts - cytology; Fibroblasts - physiology; Genome; GRB10 Adaptor Protein - deficiency; GRB10 Adaptor Protein - genetics; GRB10 Adaptor Protein - physiology; Humans; Kidney; Mice; Mice, Knockout; pharmaceutical; Proto-Oncogene Proteins c-raf - metabolism; Restriction Mapping; Signal Transduction
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M611066200

The proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.