RESUMEN
Mutations in BLM helicase predispose Bloom syndrome (BS) patients to a wide spectrum of cancers. We demonstrate that MIB1-ubiquitylated BLM in G1 phase functions as an adaptor protein by enhancing the binding of transcription factor c-Jun and its E3 ligase, Fbw7α. BLM enhances the K48/K63-linked ubiquitylation on c-Jun, thereby enhancing the rate of its subsequent degradation. Functionally defective Fbw7α mutants prevalent in multiple human cancers are reactivated by BLM. However, BS patient-derived BLM mutants cannot potentiate Fbw7α-dependent c-Jun degradation. The decrease in the levels of c-Jun in cells expressing BLM prevents effective c-Jun binding to 2,584 gene promoters. This causes decreases in the transcript and protein levels of c-Jun targets in BLM-expressing cells, resulting in attenuated c-Jun-dependent effects during neoplastic transformation. Thus, BLM carries out its function as a tumor suppressor by enhancing c-Jun turnover and thereby preventing its activity as a proto-oncogene.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Genes jun , Proteínas Proto-Oncogénicas c-jun/metabolismo , RecQ Helicasas/metabolismo , Animales , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Carcinogénesis , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Fase G1 , Células HCT116 , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Mutación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-jun/genética , RecQ Helicasas/genética , UbiquitinaciónRESUMEN
A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.
Asunto(s)
Mitosis , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencias de Aminoácidos , Cromatina/genética , Cromatina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Poro Nuclear/química , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The spectrum of tumors that arise owing to the overexpression of c-Myc and loss of BLM is very similar. Hence, it was hypothesized that the presence of BLM negatively regulates c-Myc functions. By using multiple isogenic cell lines, we observed that the decrease of endogenous c-Myc levels that occurs in the presence of BLM is reversed when the cells are treated with proteasome inhibitors, indicating that BLM enhances c-Myc turnover. Whereas the N-terminal region of BLM interacts with c-Myc, the rest of the helicase interacts with the c-Myc E3 ligase Fbw7. The two BLM domains act as 'clamp and/or adaptor', enhancing the binding of c-Myc to Fbw7. BLM promotes Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase-independent manner. A subset of BLM-regulated genes that are also targets of c-Myc were determined and validated at both RNA and protein levels. To obtain an in vivo validation of the effect of BLM on c-Myc-mediated tumor initiation, isogenic cells from colon cancer cells that either do or do not express BLM had been manipulated to block c-Myc expression in a controlled manner. By using these cell lines, the metastatic potential and rate of initiation of tumors in nude mice were determined. The presence of BLM decreases c-Myc-mediated invasiveness and delays tumor initiation in a mouse xenograft model. Consequently, in tumors that express BLM but not c-Myc, we observed a decreased ratio of proliferation to apoptosis together with a suppressed expression of the angiogenesis marker CD31. Hence, partly owing to its regulation of c-Myc stability, BLM acts as a 'caretaker tumor suppressor'.