Sci. Signal., 26 May 2009
Cell Biology Controlling Trafficking with a Ubiquitin Switch
Nancy R. Gough
Science Signaling, AAAS, Washington, DC 20005, USA
After stimulation, epidermal growth factor receptors (EGFRs) are internalized and can either be recycled back to the cell surface or directed to lysosomes for degradation. The EGFR itself is modified by ubiquitin and ubiquitin-like proteins that contribute to the trafficking of the receptor within the cell. Mosesson et al. provide evidence that human Lst2 (hLst2) serves as a negative regulator of EGFR signaling and that this activity of hLst2 is regulated by monoubiquitinylation. Lst2, both human and the worm homolog, contains a FYVE domain, which binds phosphatidylinositol 3-phosphate (PI3P), a lipid formed at the early endosome membrane. Consistent with the function of Lst2 in worms, EGFR signaling was enhanced in human KB cells in which Lst2 was depleted with siRNA. Because of the FYVE domain, hLst2 was expected to colocalize with endosomes; however, ectopically expressed hLst2 exhibited a nonvesicular cytoplasmic distribution. Analysis of cells expressing a fusion of just the FYVE domain of hLst2 to green fluorescent protein (GFP) revealed that the FYVE domain was functional because this fusion protein did localize to early endosomes in a manner dependent on PI3P production. Removal of either of two conserved regions, the Ndom and the CBox, also resulted in proteins that colocalized with markers of early endosomes, suggesting that these domains interfered with recognition of PI3P by the FYVE domain. Mass spectrometry analysis of hLst2 showed that the protein was modified by the addition of ubiquitin and by phosphorylation, and immunoblotting confirmed these posttranslational modifications. Both the Ndom and the CBox were necessary for ubiquitinylation, but the FYVE domain was dispensable. By following the trafficking and localization of either GFP-hLst2WT (wild-type) or GFP-hLst2K87R, a point mutant that could not be ubiquitinylated, the authors showed that the ubiquitinylation-defective mutant localized with markers of early endosomes. Analysis of a double point mutant, GFP-hLst2K87R/C823A, that lacked a functional FYVE domain showed that endosomal localization required the FYVE domain, suggesting that attachment of ubiquitin prevents the FYVE domain from recognizing its lipid binding partner. Binding to immobilized phosphoinositides confirmed that wild-type hLst2 isolated from transfected cells bound weakly to PI3P, whereas the ubiqutinylation-defective mutant bound strongly. The FYVE domain mutant also failed to bind. Tracking of fluorescently labeled EGF or monitoring EGFR distribution in cells expressing either hLst2WT or hLst2K87R showed that EGF and its receptor colocalized to a greater extent with the nonubiquitinated Lst2. Furthermore, cells expressing hLst2K87R showed more rapid degradation of the EGFR after stimulation with EGF and decreased stimulation of an EGF-responsive reporter gene. The authors suggest that a monoubiquitinylation cycle controls the function of hLst2 in trafficking EGFRs.
Y. Mosesson, D. Chetrit, L. Schley, J. Berghoff, T. Ziv, S. Carvalho, F. Milanezi, A. Admon, F. Schmitt, M. Ehrlich, Y. Yarden, Monoubiquitinylation regulates endosomal localization of Lst2, a negative regulator of EGF receptor signaling. Dev. Cell 16, 687–698 (2009). [Online Journal]
Citation: N. R. Gough, Controlling Trafficking with a Ubiquitin Switch. Sci. Signal. 2, ec176 (2009).
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