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Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity
Shaynoor Dramsi1, Michael P Scheid, Arpita Maiti
1Department of Medicine, University of British Columbia and Vancouver Hospital, Jack Bell Research Centre, Vancouver, British Columbia, Canada, V6H 3Z6.
Abstract:
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.
Insights
Phosphorylation of Bad protein at Ser-170 blocks its pro-apoptotic activity. Mutating this site to mimic phosphorylation prevents apoptosis, highlighting Ser-170
Area of Science:
- Cellular biology
- Molecular oncology
- Protein biochemistry
Background:
- Bad is a pro-apoptotic protein in the Bcl-2 family.
- Growth factors promote cell survival via Bad phosphorylation at Ser-112, -136, or -155.
- Cytokines induce Bad phosphorylation at a novel site, Ser-170.
Purpose of the Study:
- Identify the novel cytokine-induced phosphorylation site on Bad.
- Determine the functional role of Bad phosphorylation at Ser-170.
- Investigate how Ser-170 phosphorylation affects Bad's pro-apoptotic activity.
Main Methods:
- Site-directed mutagenesis of Bad protein.
- Analysis of pro-apoptotic function in HEK293 and COS-7 cells.
- Stable expression in cytokine-dependent MC/9 cell line.
Main Results:
- Mutation of Ser-170 to Ala increased Bad's apoptotic activity.
- Mutation of Ser-170 to Asp (mimicking phosphorylation) abolished apoptotic activity.
- S112A/S170D double mutant showed no apoptosis induction.
Conclusions:
- Phosphorylation of Bad at Ser-170 is critical for inhibiting its pro-apoptotic function.
- Ser-170 phosphorylation acts as a key regulator of Bad-mediated apoptosis.
- This finding provides insights into cell survival regulation by cytokines.