The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the eect of the simultaneous block of these receptors on the MPAinduced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive eects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not aect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the ®rst time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 ± 47.
Most eukaryotic cells show a strong preference for the transfer in vivo and in vitro of the largest dolichol-P-P-linked glycan (Glc 3Man9GlcNAc2) to protein chains over that of biosynthetic intermediates that lack the full complement of glucose units. The oligosaccharyltransferase (OST) is a multimeric complex containing eight different proteins, one of which (Stt3p) is the catalytic subunit. Trypanosomatid protozoa lack an OST complex and express only this last protein. Contrary to the OST complex from most eukaryotic cells, the Stt3p subunit of these parasites transfers in cell-free assays glycans with Man 7-9GlcNAc2 and Glc 1-3Man9GlcNAc2 compositions at the same rate. We have replaced Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue and found that the complex that is formed preferentially transfers the complete glycan both in vivo and in vitro. Thus, preference for Glc 3Man9GlcNAc2 is a feature that is determined by the complex and not by the catalytic subunit. N-glycosylation ͉ Saccharomyces cerevisiae ͉ Trypanosoma cruzi
It has been postulated that creation of Man 8 GlcNAc 2 isomer B (M8B) by endoplasmic reticulum (ER) ␣-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER ␣-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man 9 GlcNAc 2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man 9 GlcNAc 2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the ␣-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/ Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.
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