Epidermal Growth Factor Induces Ubiquitination of Eps15*
- Sanne van Delft‡§,
- Roland Govers¶,
- Ger J. Strous¶,
- Arie J. Verkleij‡ and
- Paul M. P. van Bergen en Henegouwen‡
- From the ‡Department of Molecular Cell Biology, Faculty of Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht and the ¶Department of Cell Biology, Faculty of Medicine, Institute of Biomembranes, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Abstract
Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor α, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.
Footnotes
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↵* This work was supported by Grant 17.182 (to S. v. D.) from the Life Sciences Foundation, which is subsidized by the Netherlands Organization for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 31-30-253-3349; Fax: 31-30-251-3655; E-mail: sanne{at}emsaserv.biol.ruu.nl.
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↵1 The abbreviations used are: Eps15, epidermal growth factor receptor pathway substrate clone 15; EGF, epidermal growth factor; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; PVDF, polyvinylidene difluoride.
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- Received March 7, 1997.
- Revision received March 31, 1997.
