Published online Oct 26, 2010. doi: 10.4331/wjbc.v1.i10.313
Revised: August 19, 2010
Accepted: August 26, 2010
Published online: October 26, 2010
AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).
METHODS: Immunofluorescence staining, 35S-Met/Cys metabolic labeling, Western blotting analysis, sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells. Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR). Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins. In vivo32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation, eIF2α phosphorylation, and p67 expression in normal and breast cancer cells.
RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus. In a sucrose gradient, approximately 30% of the overexpressed p67 was bound with ribosomes. p67 interacted with the kinase domain, but not the dsRNA-binding domains of PKR. Only the glycosylated p67 was associated with the ribosome, and p67 did not compete with PKR for ribosome binding. In breast cancer cells, there was increased autophosphorylation of PKR but no phosphorylation of eIF2α, compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.
CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.