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Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec H3T 1E2, Canada.
Abstract:
The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.
Insights
A novel PKR protein variant, PKRDeltaE7, inhibits protein synthesis regulation. This alternatively spliced form is found in various human tissues and may play a role in cell proliferation and cancer.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Protein kinase PKR (PKR) regulates protein synthesis and mediates interferon's antiviral effects.
- PKR phosphorylates eukaryotic initiation factor 2 alpha-subunit, controlling translation.
- Alternative splicing is a mechanism for protein diversity and regulation.
Purpose of the Study:
- To investigate alternatively spliced forms of PKR in human cells.
- To characterize the function and expression of a novel PKR variant, PKRDeltaE7.
- To explore the role of PKRDeltaE7 in cell proliferation and transformation.
Main Methods:
- Reverse transcriptase-polymerase chain reaction (RT-PCR) to detect PKRDeltaE7 expression.
- In vitro and in vivo assays to assess PKRDeltaE7's enzymatic activity and regulatory function.
- Analysis of PKRDeltaE7 expression levels in various human tissues and cell lines.
Main Results:
- A human T cell leukemia Jurkat cell line expresses an alternatively spliced PKR variant, PKRDeltaE7, lacking exon 7.
- PKRDeltaE7 retains dsRNA-binding motifs and exhibits dominant-negative inhibition of PKR autophosphorylation and eIF2α phosphorylation.
- PKRDeltaE7 is expressed in diverse human tissues, with higher levels in Jurkat cells compared to normal peripheral blood mononuclear cells.
Conclusions:
- PKRDeltaE7 represents a novel, alternatively spliced form of PKR with dominant-negative activity.
- The expression pattern of PKRDeltaE7 suggests a potential role in cell proliferation and transformation.
- Alternative splicing of PKR may serve as a mechanism for its own autoregulation, impacting cell growth control.