Three leucine-rich sequences and the N-terminal region of double-stranded RNA-activated protein kinase (PKR) are responsible for its cytoplasmic localization.
The double-stranded RNA-activated-protein kinase PKR was originally identified as a ribosomal protein that regulates protein synthesis at the translational level. While PKR locates predominantly to the cytoplasm, nuclear or nucleolar species of PKR have been detected. Here, we demonstrate that PKR possesses three leucine-rich sequences resembling nuclear export signals (NESs). Enhanced green fluorescent protein (EGFP) fused to one of these sequences and transfected in COS-1 cells exhibited predominant cytoplasmic staining, which was abrogated by a leucine to alanine substitution. In addition, Leptomycin B (LMB), an inhibitor of NES-mediated nuclear export, inhibited the cytoplasmic localization of EGFP-NES, indicating the potential activity of these stretches as NESs. Although EGFP fused to a PKR with three NES mutations still located to the cytoplasm, an additional N-terminal deletion impaired the cytoplasmic predominance, suggesting that the N-terminal region is also required for localization. These results suggest that the cytoplasmic localization of PKR is regulated by NESs as well as the N-terminal sequence.
Takizawa
T
T
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan. takizawa@inst-hsc.pref.aichi.jp
Tatematsu
C
C
Watanabe
M
M
Yoshida
M
M
Nakajima
K
K
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000502:physiology; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019892:eIF-2 Kinase; D007930:Leucine