RESUMEN
Mammalian alpha4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. alpha4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using alpha4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of alpha4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with alpha4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between alpha4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. alpha4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD-alpha4 immunocomplex. Furthermore, deletion mutants of alpha4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of alpha4, independent of the alpha4-PP2Ac binding site. Therefore, in addition to PP2Ac, alpha4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Células K562 , Chaperonas Moleculares , Fosfoproteínas/genética , Proteínas de Unión a Poli(A)/genética , Unión Proteica , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Carboxipeptidasas/metabolismo , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Animales , Carboxipeptidasas/química , Carboxipeptidasas/genética , Línea Celular Tumoral , Inducción Enzimática , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , RatasRESUMEN
(123)I-Iodo-alpha-methyltyrosine (IMT) transport into lymphomas has not been fully characterized. In rat Nb2-11C and Nb2-Sp lymphoma cell lines, linear uptake of (123)I-IMT occurred rapidly within 5-10 min. Eadie-Hoftee plots of (123)I-IMT uptake gave apparent Km's of 8.34+/-1.17 and 9.64+/-1.05 microM for Nb2-11C and Nb2-Sp cells, respectively, and involved the L and B(0,+) systems. In lymphoma-bearing rats, injected (123)I-IMT accumulated rapidly in the primary tumors but gave a low tumor-to-background ratio of 2:1. (123)I-IMT was transported rapidly into lymphoma cells both in vitro and in vivo, but low target-to-nontarget ratio may not make (123)I-IMT practical for scanning in vivo.