RESUMO
WDR54 is a member of the WD40 repeat (WDR) domain-containing protein family that was recently identified as a novel oncogene in colorectal cancer. However, the molecular mechanism of WDR54 and its functional association with other molecules related to tumor cell growth are unknown. Here, we show that WDR54 can be cross-linked by the action of transglutaminase (TG) 2, which enhances the activation of EGF receptor-mediated signaling pathway. The most carboxyl-terminal WD domain was required for cross-linking. In addition, lysine 280 in WDR54, also in this WD domain, was an important residue for both cross-linking and ubiquitination. Cross-linked WDR54 was found in vesicles aggregated at the plasma membrane. The activated EGF receptor was co-localized with this vesicle, and the internalization of the EGF receptor into the cytosol was sustained. As a result, Erk activity in response to EGF stimulation was enhanced. Furthermore, the growth of the cells lacking WDR54 expression generated by genome editing was delayed compared with that in wild-type cells. Because TG2 is also has been proposed to activate the EGF receptor-signaling and proliferation of tumor cells, WDR54 might have a functional relationship with the EGF receptor and TG2. Our study on the mechanism of biological function of WDR54 may provide rationale for the design and development of a cancer drug based on inhibiting the post-translational modification of this oncogene product.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Animais , Proteínas de Arabidopsis/fisiologia , Células COS , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Chlorocebus aethiops , Receptores ErbB/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Células HEK293 , Humanos , Fosforilação/fisiologia , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Transglutaminases/genética , Transglutaminases/fisiologia , UbiquitinaçãoRESUMO
Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Envelhecimento/genética , Proteínas ELAV/metabolismo , Leptina/genética , Proteínas Ligadas a Lipídeos/metabolismo , Obesidade/genética , Estabilidade de RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Deleção de Genes , Regulação da Expressão Gênica , Leptina/metabolismo , Proteínas Ligadas a Lipídeos/genética , Masculino , Metaboloma , Camundongos Knockout , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association with the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells.