Endogenous inhibitors of the dsRNA-dependent eIF-2 alpha protein kinase PKR in normal and ras-transformed cells.
The serine/threonine kinase PKR is activated by autophosphorylation in response to nanomolar concentrations of double-stranded RNA and other polyanions. We have previously shown that expression of an oncogenic ras gene induces an endogenous protein inhibitor of PKR activation in murine fibroblasts, as measured by a greatly reduced ability of PKR to autophosphorylate in response to double-stranded RNA in lysates from these ras-expressing cells. Immunoprecipitation of PKR away from the ras-transformed cell lysate restored the ability of PKR to become autophosphorylated. However, the autophosphorylation was no longer dsRNA-dependent. In the present work, PKR immobilized either by immunoprecipitation or by affinity precipitation on Agpoly(I) poly(C) was found to autophosphorylate in a dsRNA-independent manner when incubated in the presence of a detergent lysis buffer and ATP. When lysis buffer was replaced by cytoplasmic extract from normal or ras-transformed cells, autophosphorylation of the immobilized PKR was inhibited in the presence or absence of dsRNA, even though it could be shown that PKR remained bound and intact in the precipitate, and able to autophosphorylate if rewashed with lysis buffer. These findings suggest that PKR activation is regulated by an endogenous inhibitor in murine fibroblasts as well as by dsRNA or other polyanions.
Mundschau
L J
LJ
Boston University School of Medicine, Cancer Research Center, MA 02118, USA.
Faller
D V
DV
D000818:Animals; D002457:Cell Extracts; D002461:Cell Line, Transformed; D004800:Enzymes, Immobilized; D011905:Genes, ras; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D019892:eIF-2 Kinase; D018631:ras Proteins / Q000378:metabolism*
D002457:Cell Extracts; D004800:Enzymes, Immobilized; D012330:RNA, Double-Stranded; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D018631:ras Proteins