Characterization of a novel, cytokine-inducible carboxypeptidase D isoform in haematopoietic tumour cells.
CPD-N is a cytokine-inducible CPD (carboxypeptidase-D) isoform identified in rat Nb2 T-lymphoma cells. The prototypic CPD (180 kDa) has three CP domains, whereas CPD-N (160 kDa) has an incomplete N-terminal domain I but intact domains II and III. CPD processes polypeptides in the TGN (trans-Golgi network) but the Nb2 CPD-N is nuclear. The present study identified a cryptic exon 1', downstream of exon 1 of the rat CPD gene, as an alternative transcription start site that encodes the N-terminus of CPD-N. Western-blot analysis showed exclusive synthesis of the 160 kDa CPD-N in rat Nb2 and Nb2-Sp lymphoma cells. Several haematopoietic cell lines including human K562 myeloma, Jurkat T-lymphoma and murine CTLL-2 cytotoxic T-cells express a 160 kDa CPD-immunoreactive protein, whereas mEL4 T-lymphoma cells express the 180 kDa CPD. The CPD-immunoreactive protein in hK562 cells is also nuclear and cytokine-inducible. In contrast, MCF-7 breast cancer cells express only the 180 kDa CPD, which is mainly in the TGN. CPD/CPD-N assays using substrate dansyl-L-alanyl-L-arginine show approx. 98% of CPD-N activity in the Nb2 nucleus, whereas MCF-7 CPD activity is enriched in the post-nuclear 10000 g pellet. The K(m) for CPD-N and CPD are 132+/-30 and 63+/-9 microM respectively. Specific activity/K(m) ratios show that dansyl-L-alanyl-L-arginine is a better substrate for CPD-N than for CPD. CPD-N has an optimal pH of 5.6 (due to domain II), whereas CPD has activity peaks at pH 5.6 (domain II) and pH 6.5-7.0 (domain I). CPD and CPD-N are inhibited non-competitively by zinc chelator 1,10-phenanthroline and competitively by peptidomimetic inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The Nb2 CPD-N co-immunoprecipitated with phosphatase PP2A (protein phosphatase 2A) and alpha4 phosphoprotein. In summary, a cytokine-inducible CPD-N is selectively expressed in several haematopoietic tumour cells. Nuclear CPD-N is enzymatically active and interacts with known partners of CPD.
O'Malley
Padraic G P
PG
Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5.
Sangster
Shirley M
SM
Abdelmagid
Salma A
SA
Bearne
Stephen L
SL
Too
Catherine K L
CK
D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism*; D004790:Enzyme Induction; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats
D016207:Cytokines; D007527:Isoenzymes; D002268:Carboxypeptidases; C078874:carboxypeptidase D