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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Published on: January 7, 2013
A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis
Jung D Kim1, Kwang W Seo, Eun A Lee
1School of Biological Sciences, University of Ulsan, Ulsan, Republic of Korea.
Abstract:
Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.
Insights
A novel protein kinase C delta (PKCδ) isoform, PKCδIX, was identified. This isoform acts as a dominant-negative inhibitor, suppressing apoptosis by blocking PKCδ activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein kinase C delta (PKCδ) is crucial for cell proliferation and apoptosis.
- Caspase-mediated cleavage of PKCδ generates a catalytic fragment essential for etoposide-induced apoptosis.
Purpose of the Study:
- To identify and characterize a novel mouse PKCδ isoform.
- To investigate the functional role of the novel PKCδIX isoform in apoptosis and kinase activity.
Main Methods:
- Identification of PKCδIX via alternative splicing.
- Expression analysis of PKCδIX.
- Overexpression studies in NIH3T3 cells to assess apoptosis inhibition.
- In vitro kinase assays using recombinant PKCδIX protein.
Main Results:
- A novel PKCδ isoform, PKCδIX, was identified and found to be ubiquitously expressed.
- PKCδIX lacks key domains including the C1 domain, caspase 3 cleavage site, and ATP binding site, but retains a catalytic domain and NLS.
- Overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis.
- Recombinant PKCδIX competitively inhibited PKCδ kinase activity in vitro.
Conclusions:
- PKCδIX functions as a dominant-negative inhibitor of PKCδ.
- PKCδIX can suppress apoptosis by interfering with PKCδ kinase activity in vivo.
