Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.
Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.
Dramsi
Shaynoor
S
Department of Medicine, University of British Columbia and Vancouver Hospital, Jack Bell Research Centre, Vancouver, British Columbia, Canada, V6H 3Z6.
Scheid
Michael P
MP
Maiti
Arpita
A
Hojabrpour
Payman
P
Chen
Xianming
X
Schubert
Kathryn
K
Goodlett
David R
DR
Aebersold
Ruedi
R
Duronio
Vincent
V
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019254:Genes, bcl-2; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D014162:Transfection; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494811:BCL2L1 protein, human; C494820:Bad protein, mouse; C494812:Bcl2l1 protein, mouse; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D010768:Phosphoserine; D012694:Serine; D011065:Poly(ADP-ribose) Polymerases