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Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence
Tracie A DeVries1, Margaret C Neville, Mary E Reyland
1Department of Craniofacial Biology, School of Dentistry, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Abstract:
We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.
Insights
Protein kinase Cdelta (PKCdelta) enters the nucleus to trigger apoptosis. Nuclear import, mediated by a C-terminal NLS, is crucial for this cell death pathway, with caspase cleavage facilitating the process.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein kinase Cdelta (PKCdelta) has been previously identified as essential for mitochondrial-dependent apoptosis.
- The precise mechanisms by which PKCdelta initiates apoptosis remain incompletely understood.
Purpose of the Study:
- To investigate the role of PKCdelta nuclear import in etoposide-induced apoptosis.
- To identify the specific signals and mechanisms governing PKCdelta nuclear translocation.
Main Methods:
- Utilized etoposide treatment in cell culture models.
- Employed techniques such as transfection, site-directed mutagenesis (targeting caspase cleavage sites and nuclear localization signals - NLS), and fluorescent protein tagging (green fluorescent protein).
- Assessed protein localization, apoptosis induction, and protective effects of kinase-negative fragments.
Main Results:
- PKCdelta is imported into the nucleus of etoposide-treated cells, and this nuclear import is essential for apoptosis.
- A nuclear localization signal (NLS) in the C-terminus of PKCdelta mediates this import, a process facilitated by caspase cleavage.
- Expression of the PKCdelta catalytic fragment (CFdelta) leads to nuclear localization and apoptosis, while mutations in the NLS block these effects and prevent apoptosis. Kinase-negative CFdelta protects cells, but only if the NLS is functional.
Conclusions:
- PKCdelta nuclear import, regulated by its NLS and caspase cleavage, is a critical step for initiating apoptosis.
- PKCdelta likely regulates essential nuclear events that trigger the apoptotic cascade.
- Targeting the nuclear import or function of PKCdelta may offer therapeutic strategies for controlling apoptosis.