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p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway
1Cell Signaling Laboratory, New England Biolabs, Beverly, Massachusetts 01915, USA. tan@neb.com
Abstract:
Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.
Insights
Protein kinase C (PKC) activation promotes cell survival by indirectly phosphorylating the pro-apoptotic protein Bad. This phosphorylation, mediated by p90 ribosomal S6 kinase (RSK), inhibits Bad-induced cell death, revealing a novel PKC survival pathway.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Protein kinase C (PKC) activation is linked to cell survival, but its downstream targets and pathways remain unclear.
- The pro-apoptotic protein Bad regulates cell death, particularly in response to growth factors like interleukin-3.
- Phosphorylation of Bad at Serine 112 (Ser112) is a key event in preventing apoptosis.
Purpose of the Study:
- To elucidate the specific targets and pathways through which protein kinase C (PKC) activation promotes cell survival.
- To investigate the role of PKC in the regulation of Bad phosphorylation and its impact on apoptotic cell death.
- To identify the kinase responsible for Bad phosphorylation at Ser112 in response to PKC activators.
Main Methods:
- Treatment of cells with PKC activators (12-O-tetradecanoylphorbol-13-acetate, bryostatin) and inhibitors.
- Western blot analysis to detect phosphorylation of Bad at Ser112.
- In vitro kinase assays using purified PKC isoforms and p90 ribosomal S6 kinase (RSK) family members.
- Assessment of Bad-14-3-3 binding and cell death assays.
Main Results:
- PKC activators stimulated Bad phosphorylation at Ser112, an event blocked by PKC inhibitors but not PI 3-kinase/Akt inhibitors.
- PKC isoforms did not directly phosphorylate Bad at Ser112 in vitro, indicating an indirect mechanism.
- p90 RSK family members (RSK-2, RSK-3) were activated by phorbol ester and directly phosphorylated Bad at Ser112 both in vitro and in vivo.
- p90 RSK-mediated phosphorylation of Bad at Ser112 promoted its binding to 14-3-3 proteins and inhibited Bad-induced cell death.
Conclusions:
- p90 ribosomal S6 kinase (RSK) acts as a critical downstream effector in a protein kinase C (PKC)-dependent pathway.
- PKC activation leads to indirect phosphorylation of Bad at Ser112 via p90 RSK, thereby inhibiting apoptosis.
- This pathway highlights a novel mechanism by which PKC activation promotes cell survival through the inactivation of Bad-mediated cell death.