Epidermal Growth Factor Induces Ubiquitination of Eps15

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. 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. Eps15 1The abbreviations used are:Eps15epidermal growth factor receptor pathway substrate clone 15EGFepidermal growth factorFCSfetal calf serumDMEMDulbecco's modified Eagle's mediumPVDFpolyvinylidene difluoride. has been identified as a 142-kDa substrate of the EGF receptor (1Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar). In quiescent cells Eps15 is associated to the EGF receptor, and upon EGF stimulation this association increases dramatically (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar). In addition, Eps15 has been shown to bind to both adaptor protein-2 and clathrin (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar, 3Benmerah A. Gagnon J. Bègue B. Mégarbané B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1996; 131: 1831-1838Crossref Scopus (151) Google Scholar). Subcellular fractionation and immunolocalization studies have shown that Eps15 is present in clathrin-coated pits and vesicles but not in early endosomes (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar, 4Tebar F. Sorkina T. Sorkin A. Ericsson M. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Eps15 shares homology with the yeast proteins End3p and Pan1p. Both proteins contain multiple Eps15 homology domains, a motif proposed to mediate protein-protein interaction, and have been implicated in the endocytosis of the α-factor and lipids, respectively (5Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar, 6Wendland B. McCaffery J.M. Xiao Q. Emr S.D. J. Cell Biol. 1996; 135: 1485-1500Crossref PubMed Scopus (202) Google Scholar). epidermal growth factor receptor pathway substrate clone 15 epidermal growth factor fetal calf serum Dulbecco's modified Eagle's medium polyvinylidene difluoride. Tyrosine phosphorylation of Eps15 is transient and occurs within 2 min of EGF stimulation (1Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar). In addition, EGF stimulation results in the appearance on SDS-polyacrylamide gels of a slowly migrating band of Eps15 of approximately 150 kDa. Tyrosine kinase activity of the EGF receptor was found to be required for this apparent increase in molecular mass of Eps15 (7van Delft S. Verkleij A.J. van Bergen en Henegouwen P.M.P. NATO ASI Series. 1997; 101: 151-161Google Scholar). Expression of Eps15 cDNA in bacteria shows the presence of only the 142-kDa form, suggesting that Eps15 is undergoing an EGF-induced post-translational modification (1Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar). In this paper we investigated the nature of this post-translational modification of Eps15. We found that the appearance of the high molecular mass form of Eps15 is not due to EGF-induced hyperphosphorylation. Instead, we found that the 8-kDa increase in molecular mass was caused by monoubiquitination of Eps15. This indicates that EGF induces two different modes of post-translational modification of Eps15: tyrosine phosphorylation and ubiquitination. HER14 fibroblasts (NIH3T3 fibroblasts stably transfected with human EGF receptor cDNA) were cultured in bicarbonate buffered Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc., Paisely, UK) supplemented with 7.5% (v/v) fetal calf serum (FCS) (Life Technologies, Inc.) in a humidified atmosphere at 37 °C. Cells were grown in 60-, 100-, or 175-mm dishes (Nunc Life Technologies, Gaithersburg, MD) till 80% confluency. Cells were serum-starved in DMEM with 0% v/v FCS for 24 h before stimulation with 50 ng/ml EGF. Cells were lysed in RIPA buffer (20 mm Tris-HCl, pH 7.4, 150 mmNaCl, 0.5% Triton X-100, 0.1% SDS, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mmbenzamidine, 100 mm NaF, and 1 mmNa3VO4) at 4 °C for 5 min, scraped from the dish, and centrifuged for 5 min at 12,000 × g in an Eppendorf centrifuge. Total cell lysate samples were prepared by adding Laemmli sample buffer to the RIPA lysates. For immunoprecipitations, the RIPA lysates were incubated with 25 μl of a 1:1 suspension of protein A-Sepharose for 1 h at 4 °C and centrifuged. Supernatants were incubated with anti-Eps15 antibody (8Schumacher C. Knudsen B. Ohuchi T. Di Fiore P.P. Glassman R.H. Hanafusa H. J. Biol. Chem. 1995; 270: 15341-15347Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) for 2 h at 4 °C. Subsequently, protein A-Sepharose was added, and after a further incubation of 2 h, the immunoprecipitates were washed three times, once with RIPA-buffer, once with high salt buffer (20 mm Tris-HCl, pH 7.4, 0.5 m NaCl, and 1% Triton X-100), and finally once with low salt buffer (20 mmTris-HCl, pH 7.4, 0.15 m NaCl, and 1% Triton X-100). For alkaline phosphatase treatment, immunoprecipitates were incubated in phosphatase buffer (50 mm Tris, pH 8.5, and 1 mm EDTA) with 50 units of alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany) at 37 °C for 30 min. Heat-inactivated alkaline phosphatase was prepared by incubating the alkaline phosphatase at 95 °C for 15 min. The beads were boiled in 20 μl of Laemmli sample buffer for 5 min, and proteins were separated by 8% SDS-polyacrylamide gel electrophoresis, Western blotted onto PVDF membrane (Immobilon-P, Millipore, Bedford MA, USA), and probed with rabbit polyclonal antibodies against Eps15, mouse monoclonal anti-phosphotyrosine PY20 (Transduction laboratories, Lexington KY), or rabbit polyclonal anti-ubiquitin antibodies (antibody kindly provided by Dr. A. Ciechanover). Protein bands were visualized by Enhanced Chemiluminescence (Renaissance, DuPont NEN, Boston, MA) using peroxidase-conjugated goat-anti-rabbit or rabbit-anti-mouse immunoglobulins (Jackson ImmunoResearch, Pennsylvania, PA). To determine the N-terminal amino acid sequence of Eps15, proteins of immunoprecipitates were separated on a 8% SDS-polyacrylamide gel and transferred to a PVDF membrane. The proteins were stained with Ponceau S (Sigma, St. Louis, MO), and the Eps15 bands were cut out of the membrane, washed thoroughly with distilled water, and subjected to Edman degradation (9Edman P. Acta. Chem. Scand. 1950; 4: 283-293Crossref Google Scholar, 10Dopheide T.A.A. Moore S. Stein W.H. J. Biol. Chem. 1967; 242: 1833-1837Abstract Full Text PDF PubMed Google Scholar). High pressure liquid chromatography was used for analysis of degradation products. Inhibition of endocytosis in HER14 cells was performed by potassium depletion (11Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G.W. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar), by incubating the cells in hypertonic medium (12Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar), by acidification of the cytosol (13Sandvig K. Olsnes S. Petersen O.W. van Deurs B. J. Cell Biol. 1987; 105: 679-689Crossref PubMed Scopus (252) Google Scholar), or by an incubation of the cells at 4 °C (14Dunn W.A. Hubbard A.L. Aronson N.N. J. Biol. Chem. 1980; 255: 5971-5978Abstract Full Text PDF PubMed Google Scholar). For potassium depletion, cells were washed twice with depletion buffer (20 mm Hepes, pH 7.4, 0.14 m NaCl, 1 mm CaCl2, 1 mm MgCl2, and 1 g/l d-glucose). Subsequently, cells were incubated for 5 min with a hypotonic buffer consisting of one part depletion buffer and one part H2O. Next, the cells were incubated for a further 30 min in depletion buffer at 37 °C. Control cells were incubated with the same buffer supplemented with 10 mm KCl. Inhibition of endocytosis by hypertonic shock of cells was performed using hypertonic medium consisting of DMEM supplemented with 0.45m sucrose. Cells were washed twice with hypertonic medium before a 30-min incubation at 37 °C in this medium. For inhibition of endocytosis by acidification of the cytosol, cells were incubated for 10 min in DMEM, pH 5.0, supplemented with 10 mm acetic acid. Control cells were incubated in DMEM, pH 5.0, without acetic acid. Inhibition of endocytosis by an incubation at 4 °C was performed by placing the cells for 30 min at this temperature in DMEM-Hepes 0% FCS, supplemented with 0.1% bovine serum albumin. For all experiments, cells were serum-starved for 24 h prior to treatment with EGF. Internalization assays were performed using a protocol modified from Haigler et al. (15Haigler H.T. Maxfield F.R. Willingham M.C. Pastan I. J. Biol. Chem. 1980; 255: 1239-1241Abstract Full Text PDF PubMed Google Scholar). HER14 cells were grown on 6-well dishes to 80% confluency and incubated for 30 min with 1 ng/ml 125I-EGF in DMEM containing 20 mm Hepes, pH 7.2, and 0.1% bovine serum albumin. Nonspecific EGF binding was measured in the presence of 500-fold molar excess of unlabeled EGF. Following this incubation, the medium was removed and the cells were washed twice with ice-cold phosphate-buffered saline. Surface-bound EGF was removed by successive incubation of the cells with 0.2 m sodium acetate, pH 3.5, containing 150 mm NaCl (acid wash) for 5 and 1 min, respectively. The acid wash solution represented the surface-bound radioactivity. Internalized ligand was determined by lysis of the cells by incubation with 1 n NaOH for 5 min at 37 °C. The rate of endocytosis was expressed as the ratio of internal and surface-bound EGF. To determine the nature of the EGF-induced increase in the molecular mass of Eps15, we investigated whether this increase is caused by tyrosine phosphorylation. HER14 fibroblasts expressing the human EGF receptor were stimulated with EGF, and Eps15 was immunoprecipitated from the cell lysates. One Eps15 immunoprecipitate was treated with alkaline phosphatase, whereas two controls were either left untreated or treated with heat-inactivated phosphatase. The proteins were separated on 8% SDS-polyacrylamide gels and blotted onto PVDF membrane. After detection of tyrosine phosphorylated Eps15 by anti-phosphotyrosine antibodies, two Eps15 bands of 142 and 150 kDa were visible in untreated cells. This indicates that both forms of Eps15 are phosphorylated (Fig.1, Con). The two Eps15 bands of 142 and 150 kDa are each resolved into a tightly spaced doubled, and both forms of the doubled are tyrosyl phosphorylated. The reason for the slight difference in molecular mass is not known but could be due to differential splicing. Treatment of Eps15 with alkaline phosphatase resulted in the complete dephosphorylation of Eps15 (Fig. 1,AP), whereas treatment with heat-inactivated alkaline phosphatase did not change the phosphorylation state of Eps15 (Fig. 1,HI-AP). Reprobing the same blot with anti-Eps15 antibodies showed that irrespective of the phosphorylation state of Eps15, the 142- and 150-kDa forms were present (Fig. 1). These results demonstrate that the appearance of high molecular mass form of Eps15 is not the result of tyrosine phosphorylation. The approximate increase of 8 kDa in the modified form of Eps15 stimulated us to investigate the possible monoubiquitination of Eps15. Ubiquitin is a highly conserved protein of about 8 kDa, which is abundant in eukaryotes. Ubiquitin is found free or covalently linked via its C terminus to NH2 groups of one or more lysine residues of a variety of cytoplasmic, nuclear, and integral membrane proteins (16Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar). To investigate the monoubiquitination of Eps15, Eps15 was immunoprecipitated from HER14 cells that were either left unstimulated or stimulated with 50 ng/ml EGF. The protein samples were separated on 8% SDS-polyacrylamide gels, and the Western blot was probed with anti-Eps15 antibodies. A clear mobility shift was seen after EGF stimulation but not in unstimulated cells (Fig. 2). Subsequently, the Western blot was stripped and reprobed with anti-ubiquitin antibodies. In this case only the 150-kDa form of Eps15 was detected, demonstrating that Eps15 becomes monoubiquitinated upon EGF stimulation (Fig. 2). In addition to the appearance of the 150-kDa band, a slight staining of higher molecular mass Eps15 was detected upon EGF addition. This phenomenon was better visible upon longer exposures (data not shown). Eps15 of higher molecular mass was previously also found on Western blots containing immunoprecipitated Eps15 that were stained for phosphotyrosine residues (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar). These observations suggest that Eps15 is not only monoubiquitinated but that a minority of Eps15 may also be multiubiquitinated. To obtain further proof for the ubiquitination of Eps15, the N-terminal sequences of the 142- and 150-kDa Eps15 isoforms were determined by Edman degradation. Because ubiquitin is conjugated via its C terminus to the target proteins, the N terminus of conjugated ubiquitin is still available for Edman degradation. Sequencing of the 142-kDa form of Eps15 did not result in any signal, most probably due to N-terminal blocking. Sequencing of the 150-kDa form of Eps15 resulted in a single protein sequence (Fig. 3). Comparison of these 10 amino acids with the published sequence of bovine ubiquitin revealed that the obtained amino acids are identical to the first amino acids of ubiquitin. Comparison of this sequence with sequences in the SWISS-PROT protein data base did not reveal a relevant match with any other protein than ubiquitin. Based on both the Western blotting results and the N-terminal amino acid sequence, we conclude that Eps15 becomes ubiquitinated after stimulation of the cell with EGF. Because the increase in molecular mass of Eps15 is similar to the molecular mass of ubiquitin (8 kDa), we conclude that Eps15 becomes predominantly monoubiquitinated. Because the approximate ratio of the two Eps15 forms in EGF-stimulated cells was previously determined as 1:1, we estimate that about 50% of Eps15 becomes monoubiquitinated after stimulation of cells with EGF (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar). Both forms of Eps15 become phosphorylated on tyrosine residues (Fig. 1), which indicates that ubiquitination of Eps15 is not required for its phosphorylation. Monoubiquitination of proteins has not frequently been reported. Examples of monoubiquitination are the T cell antigen receptor (17Hou D. Cenciarelli C. Jensen J.P. Nguygen H.B. Weissman A.M. J. Biol. Chem. 1994; 269: 14244-14247Abstract Full Text PDF PubMed Google Scholar), histone H2A (18Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (122) Google Scholar), and cytochrome c (19Sokolik C.W. Cohen R.E. J. Biol. Chem. 1991; 266: 9100-9107Abstract Full Text PDF PubMed Google Scholar). The yeast α-factor receptor has recently been shown to become either mono- or diubiquitinated (20Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar). Multiubiquitination of proteins usually starts on one lysine residue (16Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar). Subsequently, this ubiquitin becomes ubiquitinated, resulting in the formation of multiubiquitin chains. Examples of multiubiquitination include cytoplasmic and proteins but also integral membrane proteins as for EGF Davis 1995; Google Scholar), growth G. C. 1987; PubMed Scopus Google Scholar, van P. A. A.L. J. 1996; PubMed Scopus Google Scholar), growth factor S. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the factor H. W. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). Protein ubiquitination has been implicated in (16Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar). The most of ubiquitination in the of proteins for degradation to the S not all ubiquitinated proteins are suggesting for ubiquitination Treatment of cells with resulted in a of ubiquitinated histone suggesting a for ubiquitination in (18Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (122) Google Scholar). has been that ubiquitination a in the of a of the factor M. J. M.S. J.M. F. F. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a for ubiquitination has been recently described for the ubiquitination of membrane of both the growth receptor and the α-factor receptor in S. have been implicated in the endocytosis of these (20Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar, van P. A. A.L. J. 1996; PubMed Scopus Google Scholar, L. Riezman H. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of Eps15 to both adaptor protein and clathrin and the presence of Eps15 in clathrin-coated pits and vesicles of cells suggest a for Eps15 in the endocytosis of the EGF receptor. To investigate the possible Eps15 ubiquitination and EGF receptor we the of EGF receptor internalization on Eps15 ubiquitination. Internalization of EGF was inhibited in different by incubation at low by potassium from the cytosol, by a hypertonic shock of the or by acidification of the These different in hypertonic shock and incubation at low temperature of (12Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar), potassium depletion the of pits (11Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G.W. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar), whereas acidification of the cytosol is to of clathrin-coated pits from the membrane (13Sandvig K. Olsnes S. Petersen O.W. van Deurs B. J. Cell Biol. 1987; 105: 679-689Crossref PubMed Scopus (252) Google Scholar). The of these conditions on EGF endocytosis was measured EGF. In cells EGF was rapidly whereas under all endocytosis conditions EGF internalization was inhibited for more than 80% of Eps15 phosphorylation revealed that in all Eps15 phosphorylated on tyrosine residues These results demonstrate that EGF receptor activity has not been by either of these was recently by and C. S. 1996; PubMed Scopus Google Scholar) that inhibition of endocytosis using a resulted in in EGF receptor substrate phosphorylation. This indicates that Eps15 phosphorylation is before the EGF receptor internalization is of Eps15 by Western blotting showed that in cells Eps15 ubiquitinated after 10 min of EGF stimulation (Fig. 5 cells were incubated and stimulated at 4 ubiquitination of Eps15 was (Fig. 5 1). The same results were obtained endocytosis was inhibited by as potassium depletion acidification of the cytosol and hypertonic shock these data show that endocytosis is inhibited Eps15 monoubiquitination is but tyrosine phosphorylation of endocytosis Eps15 monoubiquitination. HER14 cells were subjected to different to low hypertonic potassium depletion, or cytosol the cells were left unstimulated or stimulated with 50 ng/ml EGF for 10 min. from the cell lysates were separated on 8% SDS-polyacrylamide gels, and the Western blots were incubated with anti-Eps15 antibodies Eps15 was separated in a similar and the Western blots were incubated with anti-phosphotyrosine antibodies The 142-kDa band of Eps15 and the ubiquitinated 150-kDa band of Eps15 are is the possible of the monoubiquitination of Eps15. The of Eps15 ubiquitination under conditions that EGF receptor internalization indicates that either Eps15 ubiquitination is required for Eps15 endocytosis or EGF receptor endocytosis is a for Eps15 ubiquitination. The first is an to has been for the growth receptor in cells van P. A. A.L. J. 1996; PubMed Scopus Google Scholar) and the α-factor receptor in yeast (20Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar). In this case Eps15 ubiquitination could be in the early of endocytosis of the EGF receptor. inhibition of endocytosis of the α-factor receptor in yeast resulted in an ubiquitination of the receptor, which is in to the results in this paper (20Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar). results may that endocytosis is required for Eps15 ubiquitination. This that Eps15 ubiquitination occurs at a We have shown previously that Eps15 is to pits and vesicles and from early endosomes (2van Delft S. Schumacher C. Hage W. Verkleij A.J. van Bergen en Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-823Crossref PubMed Scopus (112) Google Scholar). The monoubiquitination of Eps15 could be in the of pits to the early or in the of the is that Eps15 ubiquitination could be in the of Eps15 to the S for its which is in the first described of protein ubiquitination. We for van and of The for and the Edman experiments, and and van for