Phospholipase C-delta1 contains a functional nuclear export signal sequence.
We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-delta1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999) Biochem. Biophys. Res. Commun. 254, 284-291). Herein, we demonstrate that PLC-delta1 has a functional nuclear export signal (NES) sequence in amino acid residues 164-177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-delta1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-delta1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-delta1 after treatment with leptomycin B. These results suggest that PLC-delta1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).
Yamaga
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1297, Japan.
Fujii
M
M
Kamata
H
H
Hirata
H
H
Yagisawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D004285:Dogs; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054803:Phospholipase C delta; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta