NUCLEAR-LOCALIZATION OF THE INTERFERON-INDUCIBLE PROTEIN-KINASE PKR IN HUMAN-CELLS AND TRANSFECTED MOUSE CELLS

The levels and subcellular distribution of the interferon-inducible do uble-stranded RNA-dependent protein kinase PKR have been measured in h uman Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase, Immunofluorescence of intact cells and quan titative immunoblotting of cell extracts indicate that PKR occurs in b oth the cytoplasm and the cell nucleus, with staining specifically in the nucleolus, The ratio of cytoplasmic to nuclear PKR is approximatel y 5:1 in control cells; in response to interferon treatment the protei n kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly, Analysis of individual tr ansfected cells by confocal microscopy reveals a pattern of distributi on of PKR similar to that in Daudi cells, with immunostaining of cytop lasm and nucleoli. Similar results are observed whether cells expressi ng wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the anti body used, Two-dimensional isoelectric focusing analysis of PKR in who le cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pi, These results suggest that PKR may have a role in the cell nucleus as well as the c ytoplasm and that the subcellular distribution of the protein kinase m ay be related to post-translational modifications. (C) 1995 Academic P ress, Inc.