Phosphorylation of Bad is not essential for PKB-mediated survival signaling in hemopoietic cells.
This study was designed to investigate Bad phosphorylation at several of its key regulatory Ser residues in cytokine-dependent hemopoietic cells. These studies were initiated in light of numerous studies that have reported a key role for phosphorylated Bad in preventing apoptosis. One key question is whether the survival signaling effect of the PI 3-kinase pathway is mediated by PKB phosphorylation of Bad. We confirm previous reports that if Bad is overexpressed or if active PKB is overexpressed, then the increased phosphorylation of Bad at Ser136 is apparent. However, we were unable to detect phosphorylation of endogenous Bad at Ser136 in the MC/9 mast cell line or in murine bone marrow-derived macrophages. On the other hand, phosphorylation of Bad at Ser112 and Ser155 was observed in response to IL-3 or GM-CSF, which activate the MEK/erk pathway, but not with IL-4, which activates the PI 3-kinase, but not the MEK/erk pathway, and also promotes cell survival. In contrast to previous reports, we found that ceramide had no effect on the phosphorylation status of Bad. In summary, our results suggest that Bad phosphorylation at any of the three major sites is not a required event for cytokine-dependent cell survival, and in particular, the activation of PI 3-kinase/PKB pathway can be dissociated from phosphorylation of Bad at Ser136.
Wang
S W
SW
Department of Medicine, University of British Columbia and Vancouver Coastal Health Research Institute, Jack Bell Research Centre, 2660 Oak St., Vancouver, BC, Canada V6H 3Z6.
Denny
T A
TA
Steinbrecher
U P
UP
Duronio
V
V
D000818:Animals; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D002470:Cell Survival; D002518:Ceramides / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology; D051379:Mice; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction
D002518:Ceramides; D007377:Interleukin-3; D011994:Recombinant Proteins; D012694:Serine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor