Identification and nuclear localization of a novel prolactin and cytokine-responsive carboxypeptidase D.
A full-length, PRL-inducible complementary DNA (cDNA) encoding a novel, nuclear-targeted carboxypeptidase D isoform (designated CPD-N) was identified in the rat PRL-dependent Nb2-11C and PRL-independent Nb2-Sp lymphoma cell lines by differential display. The CPD-N cDNA (3751 bp) has 99% (3582/3583) homology with rat carboxypeptidase D (CPD; 4377 bp). In comparison to the rat CPD cDNA (ORF of 4134 bp; 180-kDa protein), CPD-N was shorter by approximately 600 bases but contained 148 unique bases at the 5'-end to give an ORF of 3399 bp. RT-PCR with primers specific to the 5'-end of CPD-N or to CPD showed that the CPD-N transcript was expressed in the Nb2-11C and Nb2-Sp cells but was not detected in rat brain or lung. Conversely, the CPD transcript was expressed in rat brain but was not detected in the two Nb2 cell lines. CPD-N expression (7.5-kb messenger RNA) was stimulated by PRL (10 ng/ml) and/or by interleukin-2 (24 U/ml) in Nb2-11C and Nb2-Sp cells. Most rat tissues expressed multiple CPD transcripts (7.5, 4.1, and 2 kb). Curiously, CPD transcripts were low or undetectable in male rat liver but readily detected in female liver, suggesting that sex-specific hormone levels may regulate its expression. Indeed, CPD expression in the PRL-responsive HepG2 hepatoma and MCF-7 breast cancer cell lines was low in control cells but was markedly stimulated by PRL after 3 h. Consistent with the shorter ORF of CPD-N, Western analysis detected proteins of smaller molecular sizes of 160 kDa (abundant) and 117 kDa (weak) in the Nb2-11C cells. The Nb2-Sp cells expressed a single and abundant 117-kDa protein, implicating differential protein processing in the two cell lines. Rat CPD has been reported to colocalize with the trans-Golgi network marker TGN38. Subcellular fractionation showed predominant nuclear localization of CPD-N and trace amounts were detected in the 100,000 x g microsomal fraction after PRL treatment (4 h); in contrast, TGN38 was found only in the microsomal fraction at this time. In cells treated with PRL for 24 h, immunofluorescent confocal microscopy showed nuclear and cytoplasmic distribution of CPD-N. Cytoplasmic CPD-N colocalized with TGN-38 whereas nuclear CPD-N had a mesh-like distribution and colocalized with nuclear lamin B.
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Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. ctoo@is.dal.ca
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D000818:Animals; D001483:Base Sequence / Q000235:genetics; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003001:Cloning, Molecular; D016207:Cytokines / Q000502:physiology*; D004790:Enzyme Induction; D005455:Fluorescent Antibody Technique; D007107:Immune System / Q000166:cytology / Q000201:enzymology; D007376:Interleukin-2 / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000201:enzymology; D011388:Prolactin / Q000494:pharmacology / Q000502:physiology*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution
D016207:Cytokines; D007376:Interleukin-2; D011388:Prolactin; D002268:Carboxypeptidases; C078874:carboxypeptidase D