Effect of EGF-receptor tyrosine kinase inhibitor on Rab5 function during endocytosis.
Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5:Q79L). AG1478 inhibitor also blocked the in vitro endosome fusion in a concentration-dependent manner, and more importantly, Rab5:Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored endosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5.
Department of Biological Sciences, Florida International University, Miami, FL 33199, USA.
Ivan
I
Jozic
Samar C
SC
Saliba
M Alejandro
MA
Barbieri
D000818:Animals; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; C490451:Rin1 protein, mouse; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins