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1.
FASEB J ; 35(4): e21337, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33715220

RESUMEN

ADP-ribosylation factors (Arfs) and Arf-like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP-bound and inactive GDP-bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase-activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans-Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP-bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity-deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull-down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1-GTP in cells expressing ArfGAP1-myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome-to-TGN retrograde transport of the Shiga toxin B-subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , ADP-Ribosilación , Factores de Ribosilacion-ADP/genética , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi , Células HeLa , Humanos , Proteínas de la Membrana/genética , Transporte de Proteínas , Interferencia de ARN
2.
FASEB Bioadv ; 6(8): 276-288, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39114447

RESUMEN

Karyopherin α 2 (KPNA2, importin α1), a transport factor shuttling between the nuclear and cytoplasmic compartments, is involved in the nuclear import of proteins and participates in cellular processes such as cell cycle regulation, apoptosis, and transcriptional regulation. However, it is still unclear which signaling regulates the nucleocytoplasmic distribution of KPNA2 in response to cellular stress. In this study, we report that oxidative stress increases nuclear retention of KPNA2 through alpha serine/threonine-protein kinase (AKT1)-mediated reduction of serine 62 (S62) phosphorylation. We first found that AKT1 activation was required for H2O2-induced nuclear accumulation of KPNA2. Immunoprecipitation and quantitative proteomic analysis revealed that the phosphorylation of KPNA2 at S62 was decreased under H2O2-induced oxidative stress. We showed that cyclin-dependent kinase 1 (CDK1), a kinase responsible for KPNA2 S62 phosphorylation, contributes to the localization of KPNA2 in the cytoplasm. AKT1 knockdown increased KPNA2 S62 phosphorylation and inhibited CDK1 activation. Furthermore, H2O2-induced AKT1 activation promoted nuclear KPNA2 interaction with nucleophosmin 1 (NPM1), resulting in attenuation of NPM1-mediated cyclin D1 gene transcription. Thus, we infer that the AKT1-CDK1 axis regulates the nucleocytoplasmic shuttling and function of KPNA2 through spatiotemporal regulation of KPNA2 S62 phosphorylation under oxidative stress conditions.

3.
Clin Cancer Res ; 26(13): 3220-3229, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32156745

RESUMEN

PURPOSE: EGFR tyrosine kinase inhibitors (EGFR-TKI) benefit patients with advanced lung adenocarcinoma (ADC) harboring activating EGFR mutations. We aimed to identify biomarkers to monitor and predict the progression of patients receiving EGFR-TKIs via a comprehensive omic analysis. EXPERIMENTAL DESIGN: We applied quantitative proteomics to generate the TKI resistance-associated pleural effusion (PE) proteome from patients with ADC with or without EGFR-TKI resistance. Candidates were selected from integrated genomic and proteomic datasets. The PE (n = 33) and serum (n = 329) levels of potential biomarkers were validated with ELISAs. Western blotting was applied to detect protein expression in tissues, PEs, and a cell line. Gene knockdown, TKI treatment, and proliferation assays were used to determine EGFR-TKI sensitivity. Progression-free survival (PFS) and overall survival (OS) were assessed to evaluate the prognostic values of the potential biomarkers. RESULTS: Fifteen proteins were identified as potential biomarkers of EGFR-TKI resistance. Cadherin-3 (CDH3) was overexpressed in ADC tissues compared with normal tissues. CDH3 knockdown enhanced EGFR-TKI sensitivity in ADC cells. The PE level of soluble CDH3 (sCDH3) was increased in patients with resistance. The altered sCDH3 serum level reflected the efficacy of EGFR-TKI after 1 month of treatment (n = 43). Baseline sCDH3 was significantly associated with PFS and OS in patients with ADC after EGFR-TKI therapy (n = 76). Moreover, sCDH3 was positively associated with tumor stage in non-small cell lung cancer (n = 272). CONCLUSIONS: We provide useful marker candidates for drug resistance studies. sCDH3 is a survival predictor and real-time indicator of treatment efficacy in patients with ADC treated with EGFR-TKIs.


Asunto(s)
Biomarcadores de Tumor , Cadherinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Proteómica , Cadherinas/sangre , Línea Celular Tumoral , Cromatografía Liquida , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Terapia Molecular Dirigida , Estadificación de Neoplasias , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica/métodos , Espectrometría de Masas en Tándem , Resultado del Tratamiento
4.
Oncogene ; 39(37): 5950-5963, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32778768

RESUMEN

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2's cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.


Asunto(s)
Glicoproteínas de Membrana/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Serina Endopeptidasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Masculino , Glicoproteínas de Membrana/química , Invasividad Neoplásica , Neoplasias de la Próstata/etiología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteolisis
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