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Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase
1Boston University School of Medicine, Cancer Research Center, Massachusetts 02118.
Abstract:
The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.
Insights
Transforming ras genes block cellular antiviral enzyme activation by double-stranded RNA (dsRNA). This ras-specific inhibition of the interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) was reversible, indicating a novel regulatory mechanism.
Area of Science:
- Molecular Biology
- Virology
- Cellular Biology
Background:
- The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a key antiviral enzyme activated by dsRNA.
- Activated dsI acts as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN).
- Transforming ras genes previously shown to block PDGF and IFN-induced gene expression.
Purpose of the Study:
- To investigate the effect of transforming ras genes on dsI activity in BALB/c-3T3 fibroblasts.
- To determine if ras-specific inhibition of dsI activation occurs.
- To elucidate the mechanism underlying ras-mediated interference with dsI activation.
Main Methods:
- Utilized BALB/c-3T3 fibroblasts expressing inducible v-Ha-ras, v-mos, or v-abl genes.
- Assessed dsRNA-mediated activation of dsI.
- Investigated morphologic reversion using cAMP or Krev-1 gene.
- Analyzed dsI inhibitory activity in cell extracts.
Main Results:
- dsRNA-mediated activation of dsI was blocked in v-ras-containing cells.
- This inhibition was specific to ras and not due to the transformed phenotype.
- Dexamethasone-inducible v-Ha-ras induced the effect within 18 hours.
- Morphologic reversion restored dsI activation potential.
- Transformation by v-mos or v-abl did not affect dsI activation.
- Latent dsI levels remained unaffected by v-ras.
- A heat-sensitive inhibitory activity, functioning in trans, was identified in v-ras cells.
Conclusions:
- Transforming ras genes specifically block dsRNA-mediated activation of the dsI enzyme.
- A novel, heat-sensitive inhibitory activity associated with ras transformation interferes with dsI activation.
- This ras-specific inhibition is reversible and not a general consequence of cellular transformation.