[Trafficking pathway and affectivity on extracellular signal-regulated kinase phosphorylation of anti-tumor fusion protein of epidermal growth factor-adenovirus early region 4 open reading frame 4 protein].
OBJECTIVE
To investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase (ERK) phosphorylation.


METHODS
MDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence, and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot.


RESULTS
The fluorescent signal of fusion protein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease.


CONCLUSIONS
Internalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.
Wang
Cun-lin
CL
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing 100005, China.
Chen
Hong
H
Ma
Xiao-li
XL
Wang
Xin
X
Huang
Bing-ren
BR
D017504:Adenovirus E4 Proteins / Q000493:pharmacokinetics*; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000493:pharmacokinetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics; D014764:Viral Proteins / Q000493:pharmacokinetics*
D017504:Adenovirus E4 Proteins; C121517:E4orf4 protein, adenovirus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004815:Epidermal Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases