RESUMEN
Translocation to the nucleus of diacylglycerol kinase (DGK)- ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.
Asunto(s)
Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Señales de Localización Nuclear/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Transporte Biológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Señales de Localización Nuclear/química , Estructura Terciaria de Proteína , Células Tumorales CultivadasRESUMEN
BACKGROUND: The significance of epidermal growth factor receptor (EGFR) expression in advanced cutaneous squamous cell carcinoma (SCC) of the head and neck remains poorly understood. METHODS: We performed a retrospective review of patients with advanced-stage (stage III or stage IV) cutaneous SCC of the head and neck (n = 56). RESULTS: The majority of patients (91%) had stage III disease, with 54% having regional metastasis and 9% with distant metastasis. Two-year survival was 64% and the 5-year survival was 56%. EGFR was found to be overexpressed in 56% of primary tumors and 58% of regional metastatic disease. Overall survival did not correlate with EGFR (p = .47) expression in primary lesions, nor was it associated with an increase in regional (p = .74) or distant metastasis (p = .56). Furthermore, there was no correlation between clinicopathologic characteristics and EGFR expression CONCLUSIONS: These data do not suggest upregulation of EGFR is associated with poor survival or aggressive disease.