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Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK
Lindsey A Allan1, Nick Morrice, Suzanne Brady
1Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK.
Abstract:
Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.
Insights
The ERK MAPK pathway inhibits apoptosis by directly phosphorylating and inactivating caspase-9. This phosphorylation blocks caspase-3 activation, promoting cell survival and potentially contributing to cancer when the pathway is overactive.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Pro-apoptotic signals activate caspase-9, initiating programmed cell death.
- Survival signals can inhibit apoptosis, but the molecular targets are often unknown.
- The Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is linked to cell survival and inhibition of apoptosis.
Purpose of the Study:
- To investigate the mechanism by which the ERK MAPK pathway inhibits apoptosis.
- To identify the specific target of ERK MAPK pathway-mediated inhibition of caspase activation.
Main Methods:
- Utilized mammalian cell extracts and biochemical assays.
- Investigated the role of protein phosphatases and kinases in caspase activation.
- Employed kinase inhibitors (staurosporine, MEK1/2 inhibitors) and specific MAPK activators (EGF, TPA).
- Performed in vitro phosphorylation assays using ERK2 and identified phosphorylation sites on caspase-9.
Main Results:
- Caspase-9 and caspase-3 activation is dependent on okadaic-acid-sensitive protein phosphatase activity.
- ERK MAPK pathway activation inhibits caspase-9 activity through direct phosphorylation.
- Caspase-9 is phosphorylated by ERK2 at Thr 125 in a MEK-dependent manner.
- Phosphorylation at Thr 125 prevents caspase-9 processing and subsequent caspase-3 activation.
Conclusions:
- The ERK MAPK pathway directly inhibits apoptosis by phosphorylating and inactivating caspase-9.
- This mechanism promotes cell survival during development and tissue homeostasis.
- Constitutive activation of the ERK MAPK pathway may contribute to tumorigenesis by inhibiting apoptosis.