Grb10 and active Raf-1 kinase promote Bad-dependent cell survival.
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.
Kebache
Sem
S
Biotechnology Research Institute, National Research Council, Montreal (PQ), Canada.
Ash
Josée
J
Annis
Matthew G
MG
Hagan
John
J
Huber
Maria
M
Hassard
Jennifer
J
Stewart
Colin L
CL
Whiteway
Malcolm
M
Nantel
André
A
D000230:Adenocarcinoma; D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D051383:GRB10 Adaptor Protein / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D016678:Genome; D006801:Humans; D007668:Kidney; D051379:Mice; D018345:Mice, Knockout; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015183:Restriction Mapping; D015398:Signal Transduction; D051025:bcl-Associated Death Protein / Q000502:physiology*
C494820:Bad protein, mouse; C495959:Grb10 protein, mouse; D051025:bcl-Associated Death Protein; D051383:GRB10 Adaptor Protein; D019908:Proto-Oncogene Proteins c-raf