extfieldsofstudy: Medicine Biology

text:
Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells.

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (approximately 50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.

family including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAP kinases mediate cell growth, adaptation, and differentiation, in part, by phosphorylating and activating transcription factors that regulate genes required for execution of these programs (1). The PC12 pheochromocytoma cell line (2) provides a useful cell culture system in which to explore the mechanism by which MAP kinases control cell differentiation. Incubation of PC12 cells with nerve growth factor (NGF) leads to growth arrest and cellular hypertrophy as well as the acquisition of a neural morphology typified by extension of neurites. A molecular correlate of the morphologic differentiation of PC12 cells is induction of neurofilaments (3) that are composed of heavy, medium, and light polypeptide chains (4). Increased expression and phosphorylation (3,5) of neurofilament proteins is thought to contribute to the extension and stabilization of neuronal processes. Moreover, the rat NFLC promoter has been characterized and shown to be induced by NGF in PC12 cells (6), indicating that transcriptional induction contributes to the increased content of neurofilament proteins.
Mutational analysis of the NGF receptor has defined the requirement for collaborative signaling through phospholipase C␥ (PLC␥) and the monomeric G protein, Ras, for morphologic differentiation of PC12 cells (7,8). Similar mutational studies with ectopically expressed ␤PDGF receptors demonstrated the requirement for Ras signaling in combination with signaling through the Src family tyrosine kinases or regulation of PLC␥ (9,10). A clearly defined action of Ras within cells is the stimulation of MAP kinase cascades including the ERK pathway (11). The ability of constitutive active forms of proximal regulators of the ERK pathway to induce morphological differentiation suggests that the ERK pathway is necessary and perhaps sufficient for morphologic differentiation of PC12 cells (12). However, mutant ␤PDGF receptors that can activate the ERK pathway, but not Src or PLC␥, failed to induce morphologic PC12 cell differentiation (10), indicating that the ERK pathway is not sufficient for growth factor-induced PC12 cell differentiation and requires collaboration with other signals. Expression of GTPase-deficient, constitutive active forms of G␣ q proteins or incubation with bone morphogenetic protein-2 induced PC12 cell differentiation accompanied by persistent activation of the JNK family of MAP kinases (13,14). Significantly, the ERKs were not activated in these settings, indicating that PC12 cell differentiation can sometimes occur independently of the ERK pathway and that the JNK pathway may contribute as well to transcriptional induction of PC12 cell differentiation.
In this study, we have employed a luciferase reporter construct controlled by the neurofilament light chain (NFLC) promoter to define MAP kinase pathways required for regulation by NGF. Our findings indicate that the ERK pathway functions in a collaborative fashion with the JNK pathway to regulate the NFLC promoter. In addition, our studies show that these MAP kinases integrate at a cAMP-response element/activating transcription factor enhancer site that binds the transcription factors c-Jun, JunD, and CREB to regulate the NFLC promoter in response to NGF.

Materials
PD098059 and SB203580 were purchased from Biomol (Plymouth Meeting, PA). NGF, EGF, and IGF-1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The neurofilament light chain antibody was purchased from Sigma. Antibodies to CREB and phospho-S73 c-Jun were from New England Biolabs (Beverly, MA), and the antibody to c-Jun and JunD was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Expression Vectors and Reporters
NFLC-Luciferase Reporters-Based on the reported sequence of the rat NFLC promoter (6), the forward primer 5Ј-GTAGCGGTACCCGTC-CCCTATGTATTGAGA-3Ј and reverse primer 5Ј-CCTCAGGCCCGGG-GCGGAGATAGGGGACCT-3Ј were synthesized and used to amplify by PCR the NFLC promoter sequences Ϫ655 to ϩ82 from rat PC12 cell genomic DNA. The PCR product was cloned into the pT7 Blue (Novagen) vector and submitted to DNA dideoxy sequencing, revealing a sequence identical to that deposited under the GenBank TM accession number X53981 and previously reported by Reeben et al. (6). The NFLC promoter was excised from pT7 Blue with KpnI (cuts at Ϫ646 bp of the promoter) and HindIII (cuts in the polylinker of pT7 Blue) and ligated into pA3Luc, a promoterless luciferase vector, that had been doubledigested with KpnI and HindIII, thereby generating NFLC-Luc. The CRE/ATF site at Ϫ46 to Ϫ36 bp (6) was converted from CTGCGTCA-GAG to CCAAATCAGAG (CRE-Mut) within the Ϫ650 to ϩ82 NFLC promoter by PCR-based mutagenesis.
Expression Vectors for c-Jun and c-Jun-DBD-An HindIII-NotI fragment of human c-JUN cDNA was excised from pRSV-c-Jun and ligated into pLNCX digested with HindIII and HpaI. Sequences encoding the DNA-binding domain of c-Jun (residues 224 -331) were amplified from the human c-JUN cDNA and epitope-tagged (hemagglutinin) by PCR, sequenced, and ligated into the HindIII and ClaI sites of pLNCX.

Transient Transfections
Aliquots of PC12 cells (2 million cells in 100 l) were electroporated (220 V and 250 microfarads with a GeneZAPPER (IBI, New Haven, CT)) in Gene Pulser 0.4-cm electrode gap cuvettes (Bio-Rad). PC12 cells were transfected with 2 g of NFLC-Luc, 1 g of pCMV-␤Gal for determination of transfection efficiency, 10 g of salmon sperm DNA as a carrier, and other expression plasmids as indicated in the figure legends. Following electroporation, the PC12 cells were plated on collagen-coated dishes in full media for 24 h at 37°C. The growth medium was subsequently replaced with DMEM containing 1% horse serum and antibiotics with growth factors or protein kinase inhibitors as indicated in the figure legends, and the transfected cells were returned to the incubator for 3 days. The transfected PC12 cells were then collected, washed once with ice-cold phosphate-buffered saline, and resuspended in 250 l of Luciferase Reporter Lysis Buffer (Promega). The cell lysates were centrifuged in a microcentrifuge, and aliquots (80 l) of the supernatants were assayed for luciferase activity with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) and luciferase assay substrate from Promega. Aliquots (15 l) of the extracts were also assayed for ␤-galactosidase (␤-Gal) to correct for transfection efficiency. The data are presented as relative light units (RLU) per milliunit of ␤-galactosidase (RLU/milliunit ␤-Gal).
Overlapping sense and antisense oligonucleotides encompassing the CRE/ATF site located at Ϫ56 to Ϫ28 within the NFLC promoter sequence (6) (5Ј-CGATCACAGTCTGCGTCAGAGT-3Ј and 5Ј-CGCCGGG-ACTCTGACGCAGACTGTGAT-3Ј) or the CRE-Mut (5Ј-CGATCACAG-TCCAAATCAGAGT-3Ј and 5Ј-CGCCGGGACTCTGATTTGGACTGTG-AT-3Ј) sequences were annealed, the 5Ј-extensions filled with DNA polymerase (Klenow fragment) and deoxynucleotide triphosphates containing [␣-32 P]dCTP, and purified by gel filtration. Aliquots (80,000 cpm) of the 32 P-labeled oligonucleotides were incubated (30 min, 4°C) in a total volume of 20 l with 5 g of nuclear extracts, 0.125 g of poly(dI-dC) in 20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, and 20% glycerol. For competition studies, the indicated concentrations of unlabeled double-stranded oligonucleotides were added to the reaction prior to addition of the radiolabeled probe. In the supershift experiments, antibodies were preincubated at 4°C for 30 min prior to the addition of 32 P-labeled DNA. Protein-DNA complexes were electrophoresed on 5% acrylamide gels at 25 mA/gel for 2 h in 25 mM Tris, 1.0 mM EDTA, 190 mM glycine. The gels were dried and exposed to x-ray film for 12-24 h.

RT-PCR Analyses
RNA was isolated with TRIzol reagent (Invitrogen) from PC12 cells that had been cultured for 1-4 days in DMEM containing 1% horse serum with or without 100 ng/ml NGF. Reverse transcription of total RNA (5 g) was performed in a volume of 20 l using random hexamers and murine leukemia virus-reverse transcriptase according to the man-ufacturer's protocol (PerkinElmer Life Sciences). Aliquots (10 l) of the reverse transcription reactions were then submitted to PCR with oligonucleotides specific for the rat NFLC mRNA (forward primer, 5Ј-TCAC-CAGCGTGGGTAGCATAAC-3Ј; reverse primer, 5Ј-TGGGGCTCAAT-CTTTCTTCTTAGC-3Ј) yielding a 436-bp product or specific for rat GAPDH mRNA (forward primer, 5Ј-ATCACCATCTTCCAGGAGCGAG-3Ј; reverse primer, 5Ј-CAACGGATACATTGGGGGTAGG-3Ј) yielding a 508-bp product. The PCR conditions for amplification of NFLC were 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min, and the GAPDH PCR conditions were 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. Aliquots (20 l) of the PCRs were electrophoresed on 1.5% agarose gels, stained with ethidium bromide, and photographed.
To confirm that the NFLC PCR amplified authentic NFLC cDNA, a Southern blot was performed. The agarose gel containing the NFLC PCR products was denatured for 30 min in 0.2 N NaOH, 0.5 M NaCl and neutralized by two washes in 50 mM Tris acetate (pH 7.8), 2.5 mM EDTA followed by one wash in 10 mM Tris acetate (pH 7.8), 0.5 mM EDTA. The gel was electrophoretically transferred to a Zeta-Probe (Bio-Rad) membrane, UV cross-linked, and pre-hybridized at 42°C in 10 ml of Rapid-hob buffer (Amersham Biosciences). A single-strand oligonucleotide probe (5Ј-TCAGAGGTGGAGGAGACCATTG-3Ј) corresponding to NFLC base pairs 1428 -1449 was labeled with T4 polynucleotide kinase and [␥-32 P]ATP and incubated with the membrane at a concentration of 1 ϫ 10 6 cpm/ml for 1 h at 42°C. The membrane was subsequently washed at room temperature in 5ϫ SSC, 0.1% SDS and twice in 1ϫ SSC, 0.1% SDS at 42°C and exposed to x-ray film for 2 h.

Assay of JNK and ERK Activity
PC12 cells were lysed in MAP kinase lysis buffer and clarified by a 5-min microcentrifugation (10,000 ϫ g). For assay of JNK activity, aliquots of the extracts containing 200 g of protein in a volume of 0.5 ml were incubated for 2 h at 4°C with 1 g of anti-JNK1 (JNK1 (C-17) from Santa Cruz Biotechnology, Santa Cruz, CA) and 10 l (packed bead volume) of protein A-Sepharose. The complexes were washed three times by repetitive centrifugation in lysis buffer and then incubated for 20 min at 30°C in 40 l of 50 mM ␤-glycerophosphate (pH 7.2), 0.1 mM sodium vanadate, 10 mM MgCl 2 , 100 M [␥-32 P]ATP (ϳ5,000 cpm/pmol) containing 2 g of His 6 -ATF2-NT as described previously (20). The reactions were terminated with 10 l of SDS-PAGE sample buffer and submitted to 10% SDS-PAGE. The ATF2-NT polypeptides were identified in Coomassie-stained gels, excised, and counted in a scintillation counter. ERK activity was similarly assayed with an immune complex assay using 2 g each of anti-ERK1 (C-16, Santa Cruz Biotechnology) and anti-ERK2 (C-14, Santa Cruz Biotechnology), and the peptide substrate EGFR-(661-682) as described previously (13).

Induction of NFLC mRNA and Protein by NGF in PC12
Cells-The NFLC protein is a defined marker of neural cells that is involved in neurite outgrowth and function (4). In this study, we explored whether the NFLC gene is a target of NGF-stimulated MAP kinase pathways in PC12 cells. As determined by RT-PCR, the NFLC mRNA was induced in a delayed fashion with a maximal observed increase of ϳ10-fold after 3-4 days of NGF treatment (Fig. 1, A and B). This finding is consistent with a previous report (21) of increased NFLC transcription and mRNA accumulation in NGF-treated PC12 cells. The content of the GAPDH mRNA did not change over the same time course (Fig. 1C). Similar to the delayed induction of NFLC mRNA, the NFLC protein as detected by immunoblot analysis was increased by 2 days and maximally increased after 3-4 days of NGF treatment. This time course of NFLC mRNA and protein accumulation coincides with the onset of neurite outgrowth in NGF-stimulated PC12 cells.

Stimulation of the NFLC Promoter by Growth Factors Is Correlated with Their Activity as Differentiation Factors and
Dependent on Ras-We constructed a luciferase reporter containing NFLC promoter sequences Ϫ655 to ϩ82 (see "Experimental Procedures") to identify signal pathways that mediate NGF-stimulated NFLC induction as a marker for PC12 cell differentiation. Transient transfection of the NFLC-Luc plasmid into PC12 cells revealed a pronounced ϳ8-fold induction of luciferase activity (Fig. 2) following 3 days of incubation with NGF, similar to the degree of NFLC mRNA induction observed in Fig. 1. A weaker ϳ3.5-fold induction of NFLC-Luc activity was measured in response to EGF, consistent with the relative ability of these two growth factors to induce morphologic differentiation in our PC12 cell cultures. IGF-1, a potent trophic factor but inactive differentiative agent for PC12 cells (22), failed to stimulate NFLC-luciferase over the basal activity and also failed to increase NFLC protein content (Fig. 2, inset).
Morphologic differentiation of PC12 cells by NGF requires signal transduction through Ras, and expression of constitutive active Ras elicits many of the morphologic and molecular actions of NGF in PC12 cells. Expression of constitutive active Ha-Ras (RasG12V) stimulated the NFLC promoter activity 4.5-fold from 144 Ϯ 30 to 654 Ϯ 64 RLU/milliunit ␤-Gal, similar to the 6-fold NGF response (850 Ϯ 157 RLU/milliunit ␤-Gal) in these experiments. By contrast, transfection of the dominant-negative Ras molecule, RasN17, yielded basal and NGF-stimulated luciferase activities of 252 Ϯ 81 and 422 Ϯ 118 RLU/milliunit ␤-Gal, respectively, a significantly reduced 1.7-fold stimulation. Thus, the growth factor selectivity (NGF Ͼ EGF Ͼ IGF-1) and Ras requirement of NGF-stimulated NFLC-luciferase activity are consistent with the previously established growth factor sensitivity and Ras requirement of PC12 cell differentiation. PC12 cells were treated for 0 -4 days with NGF, and total RNA was prepared as described under "Experimental Procedures." A, aliquots (5 g) of total RNA were submitted to RT-PCR analysis using primers specific for NFLC. Portions of the PCRs were resolved by agarose gel electrophoresis and stained with ethidium bromide. B, the agarose gel shown in A was Southern-blotted with a 32 P-labeled oligonucleotide specific for NFLC. The washed filter was exposed to x-ray film for 2 h. C, aliquots of total RNA were submitted to reverse transcriptase-PCR analysis using primers specific for GAPDH, and the products were resolved by agarose gel electrophoresis. D, PC12 cells were incubated with NGF for the indicated number of days and dissolved in MAPK lysis buffer to generate a whole-cell extract (see under "Experimental Procedures"). Aliquots containing 100 g of total cellular protein were resolved by SDS-PAGE and immunoblotted with an NFLC antibody.

Evidence for Collaboration of the JNK and ERK MAP Kinases in NGF-stimulated NFLC-Luciferase Induction in PC12
Cells-Ras is a proximal regulator of multiple signal pathways including the ERK and JNK MAPK pathways (11). Transfection of constitutive active c-Raf (BXB-Raf), an upstream activator of the ERK pathway, significantly increased NFLC-Luc activity in the absence of NGF (Fig. 3, A and B). The BXB-Rafstimulated NFLC-Luc activity was completely inhibited by the MEK inhibitor, PD098059 (Fig. 3A), or co-transfected kinaseinactive MEK1 (MEK1-K97M) (Fig. 3B). However, a concentration of PD098059 or MEK1-K97M sufficient to inhibit completely the BXB-Raf response only partially (ϳ40 -50%) inhibited NGF-induced NFLC-Luc activity (Fig. 3, A and B). This is consistent with the reduction, but not elimination, of the NGF-stimulated neurite outgrowth response in our PC12 cell cultures by PD098059 (not shown). The partial reduction of NFLC-Luc induction by PD098059 was not due to a partial MEK inhibition in these experiments because the concentration of drug employed completely inhibited persistently increased ERK activation measured after 24 h of NGF treatment from 3.6-fold in control cells to 0.8-fold in cells treated with PD098059. Thus, two different ERK pathway inhibitors at levels sufficient to inhibit the Raf-1 response yielded only a partial inhibition of the NGF stimulation, indicating that additional signal pathways collaborate with the ERK pathway for full regulation of the NFLC promoter by NGF.
The p38 MAPK pathway has been reported to be regulated by NGF in PC12 cells and also proposed as a signal pathway that mediates some of the actions of NGF in PC12 cells (23). Expression of a constitutive active MKK6 cDNA in which the activation-associated phosphorylation sites are mutated to glutamic acid (MKK6-EE) significantly increased NFLC-Luc activity from 125 Ϯ 26 to 671 Ϯ 109 RLU/milliunit ␤-Gal (p Ͻ 0.05), indicating that the p38 MAPK pathway can activate this NGF-responsive promoter. However, incubation of PC12 cells with the 5 M SB203580, a p38 MAPK inhibitor, significantly (p Ͻ 0.05) reduced the MKK6-EE-stimulated luciferase activity (206 Ϯ 34 RLU/milliunit ␤-Gal) but had no significant effect on the induction of the promoter by NGF (1887 Ϯ 497 and 1787 Ϯ 607 RLU/milliunit ␤-Gal for control and SB203580-treated, respectively). Thus, the p38 MAPK pathway does not significantly contribute to regulation of the NFLC promoter by NGF.
Transfection of an activated form of MEKK1 also significantly stimulated NFLC-Luc activity but was only modestly reduced by PD098059 (Fig. 3A), indicating that MEKK1 activated the NFLC promoter largely through MEK/ERK-independent pathways. MEKK1 is a known upstream activator of the JNK pathway (24), suggesting that MKK4 or MKK7, proximal activators of the JNK pathway, may mediate the increased NFLC-Luc activity. Although gain-of-function forms of MKK4 or MKK7 analogous to constitutive active MEK1 and MKK6 do not exist, a fusion protein of MKK7 and JNK1 exhibits a gain-of-function phenotype when transfected into cells and activates neither ERKs nor p38 MAPKs (15). We generated a similar construct with an N-terminal MKK7 cDNA fused in frame with a C-terminal JNK3 cDNA and observed constitutive activity as assessed in vivo with c-Jun-GAL4 transcriptional activation assays and in vitro with c-Jun protein kinase activity (data not shown). As shown in Fig. 4A, transfection of the MKK7-JNK3 construct alone activated the NFLC-Luc reporter ϳ3-fold. Moreover, co-transfection of MKK7-JNK3 and BXB-Raf yielded a more than additive response compared with ei- FIG. 2. Induction of neurofilament light chain promoter activity and protein by growth factors in PC12 cells. PC12 cells (2 ϫ 10 6 cells) were transfected by electroporation with 2 g of NFLC-Luc plasmid. The next day, the cells were fed with DMEM containing 1% horse serum with or without 100 ng/ml NGF, 10 ng/ml EGF, or 100 ng/ml IGF-1 and cultured for an additional 3 days. Cells were then collected and assayed for luciferase and ␤-Gal (to monitor transfection efficiency) with standard assays. Inset, PC12 cells were cultured for 4 days in the presence or absence of NGF or IGF-1, and cell extracts were prepared with MAP kinase lysis buffer (13). Aliquots of whole-cell lysates containing 100 g of total cellular protein were resolved by SDS-PAGE and submitted to immunoblot analysis with a commercially available antibody to NFLC. To test directly the role of the JNK pathway in regulation of the NFLC promoter by NGF, an expression vector encoding a kinase-inactive form of MKK7 (MKK7-KM) was co-transfected with NFLC-Luc. MKK7-KM inhibited NGF-stimulated NFLCluciferase activity ϳ30% (Fig. 4B), similar to the reduction achieved by MEK1-KM (Fig. 3B). Expression of both MKK7-KM and MEK1-KM produced an additive inhibition of NFLC-luciferase (Fig. 4B), indicating a collaboration of the ERK and JNK pathways in the induction of the NFLC promoter by NGF. A similar additive inhibition was observed with kinase-inactive MKK4 and MEK1-KM (data not shown). Note that the maximal inhibition of NGF-induced NFLC promoter induction by kinase-inactive MEK1 and MKK7 was ϳ60%. It is possible that MKK7-KM is unable to inhibit completely signaling through the JNK pathway. However, simultaneous transfection of kinase-inactive forms of MKK7 and MKK4 did not yield a greater degree of inhibition than either alone (data not shown), and MEK1-KM is able to inhibit completely the induction of NFLC-Luc by BXB-Raf (Fig. 3B). Thus, an alternative explanation is that signal pathways in addition to the JNKs and ERKs that are presently undefined also integrate at the NFLC promoter for full regulation by NGF.
The findings in Figs. 1-4 confirm the NFLC promoter responsiveness to NGF previously shown (6) and indicate that the JNKs as well as the ERKs integrate to mediate the stimulatory action of NGF. Based on these findings, persistent activation of the JNK pathway as well as the ERK pathway by NGF in PC12 cells is predicted. We measured JNK activity in PC12 cells treated for 10 min or 3 days with NGF using a specific JNK-IP assay (see "Experimental Procedures"). The results revealed an ϳ2and 8-fold increase in JNK activity following a 10-min or 3-day NGF treatment, respectively. In the same extracts, ERK activity was increased 27-and 6-fold, respectively. The modest increase in JNK activity after brief exposures to NGF and the more robust increases in JNK activity observed after several days of NGF treatment are consistent with findings in our previous publications (13,25) as well as those of Malek et al. (26). Thus, the requirement for the ERK and JNK pathways in NFLC regulation by NGF is consistent with their persistent activation by this neurotrophin.

Identification of Promoter Sequences and Cellular Transcription Factors Involved in NGF Regulation of the NFLC Promoter-
The NFLC promoter contains multiple predicted enhancer sites with unknown requirements for NGF regulation (6). As shown in Fig. 5, analysis of the NFLC promoter sequence with the MacVector (Oxford Molecular Group) transcription factor enhancer site data base identified an AP1-TRE site, several predicted Sp1 sites, a CCAAT/enhancer-binding protein (C/ EBP) site, an NFB site, an EGR-1 site, and a CRE/activating transcription factor (ATF)-like site. A recent study has highlighted C/EBP␤ as a neuronal transcriptional regulator that is activated by NGF signaling (27). Ziff and co-workers (28) have identified the Sp1 transcription factor-binding sites in the cyclin D and p21 WAF promoters as putative NGF-responsive regulatory elements. Finally, the AP1 and CRE/ATF-binding sites in the NFLC promoter are elements known to integrate MAPK signaling pathways (1). To begin to identify the sequences within the NFLC promoter that are involved in mediating induction by NGF, we generated NFLC promoter-luciferase constructs progressively truncated at the 5Ј end (Ϫ401, Ϫ201, Ϫ128, Ϫ98, Ϫ38, and Ϫ19 bp) and containing sequences up to position ϩ82 base pairs. As shown in Fig. 5, deletion of sequences up to Ϫ128, which removes the predicted Sp1 sites, did not significantly influence the induction of the promoter by NGF. Further truncation to Ϫ98 bp markedly reduced the induction of luciferase activity by NGF and truncation to Ϫ38 bp, which deletes the CRE/ATF site, completely abolished NGF induction of the NFLC promoter, and yielded a basal activity similar to that observed with the TATA-less Ϫ19 bp promoter. Thus, NFLC promoter sequences between Ϫ128 and Ϫ38 are necessary for NGF-dependent regulation of the NFLC promoter in PC12 cells, and the CRE/ATF site at Ϫ45 to Ϫ39 bp is highlighted as one putative enhancer site that may integrate the response.
EMSA were performed with an EMSA oligonucleotide that flanks NFLC promoter sequences from Ϫ60 to Ϫ28 as well a mutant oligonucleotide in which a 4-base pair change was introduced into the CRE/ATF site (in bold), see Fig. 6. The wild-type oligonucleotide probe was bound by proteins present in nuclear extracts prepared from control and NGF-stimulated PC12 cells (Fig. 6A). The binding was specific as defined by complete competition of the binding by inclusion of unlabeled (cold) oligonucleotides. A greater amount of binding in extracts from NGF-stimulated cells was consistently observed relative to extracts prepared from control cells. Analysis of nuclear proteins that bound to the mutant probe, which is predicted to abolish CREB binding, failed to exhibit specific binding of nuclear proteins in either extract. To determine whether the CRE/ATF site is important for NGF-dependent regulation of the NFLC promoter in PC12 cells, the mutant CRE/ATF sequence was introduced into the Ϫ655/ϩ82 NFLC-Luc construct, and the ability of NGF to induce luciferase activity following transfection of PC12 cells was assessed. As shown in Fig. 6B, introduction of the mutant CRE/ATF sequence into the NFLC promoter reduced NGF stimulation of the reporter by ϳ80%. This result supports the findings in Fig. 5, which indicate that the CRE/ATF sequence within the NFLC promoter is a necessary enhancer element involved in NFLC regulation by NGF in PC12 cells. However, the CRE/ATF site is probably not sufficient for NGF induction of the NFLC promoter since the NFLC promoter truncated to Ϫ98 bp exhibits markedly reduced induction by NGF.
We used EMSA coupled with antibody supershift analysis to identify transcription factors in PC12 cell nuclear extracts that bind the CRE/ATF sequence at Ϫ45 to Ϫ39 bp of the NFLC promoter. As shown in Fig. 7, inclusion of a CREB antibody in the assay resulted in the supershift of the majority of the complex detected with the CRE/ATF probe in extracts from control and NGF-treated PC12 cells. However, a residual amount of complex reproducibly remained un-shifted with the CREB antibody in extracts from NGF-stimulated cells, indicating that a factor distinct from CREB contributes to the binding of EMSA probe in extracts from NGF-treated cells. The c-Jun antibody shifted a minor amount of the complex formed with extracts from control cells and a significantly greater amount of complex in extracts from NGF-treated cells. Likewise, a JunD antibody induced a diffuse supershift of the complex detected in extracts from NGF-treated cells but not control cells. By contrast, antibodies to ATF2 and JunB failed to induce a supershift of the complexes detected with EMSA probes (data not shown). Combined, the data in Figs. 6 and 7 indicate that CREB, c-Jun, and JunD are transcription factors that interact with the CRE/ATF site in the NFLC promoter and that c-Jun and JunD are recruited into this complex following stimulation with NGF.
To determine whether the appearance of JunD in the EMSA FIG. 5. Progressive truncation of the NFLC promoter defines sequences required for induction by NGF. A linear depiction of the NFLC promoter with a number of known enhancer sites is shown. The indicated NFLC promoter fragments were generated by PCR and cloned into the pA3Luc vector, and the resulting reporters were transfected into PC12 cells. Following a 3-day incubation in the absence or presence of NGF, luciferase and ␤-Gal activities were measured.
FIG. 6. The CRE/ATF sequence in the NFLC promoter binds PC12 cell nuclear proteins and is required for NGF-induced activation. A, nuclear extracts were prepared from control (C) and NGF-treated (N) PC12 cells and tested in EMSA experiments in the presence or absence of unlabeled (cold) oligonucleotide with wild-type and mutant DNA probes flanking the CRE/ATF sequence in the NFLC promoter. The sequences of the wild-type and CRE-Mut oligonucleotides are shown. B, PC12 cells were transfected with wild-type NFLC-Luc or mutant promoters where the CRE/ATF enhancer site was mutated to the sequences corresponding to CRE-Mut. The cells were incubated for 3 days with or without NGF, and luciferase and ␤-Gal activities were measured. complex from NGF-treated cells was due to induction of this transcription factor by NGF, extracts were prepared from PC12 cells treated for 0 -4 days with NGF and immunoblotted for c-Jun, CREB, and JunD (Fig. 8A). As shown, JunD was highly expressed in control PC12 cells and only modestly induced (less than 2-fold) in response to NGF. Thus, the recruitment of JunD to the CRE/ATF binding complex was not due to induction of the JunD protein. Likewise, the cellular content of CREB was not altered following NGF treatment. By contrast, c-Jun protein was markedly and persistently induced by NGF, consistent with previous reports (29,30). Moreover, phosphorylation of c-Jun at Ser-73, as assessed by immunoblotting with a phospho-Ser-73-specific antibody, was stably increased (Fig. 8A), consistent with the persistent JNK activation in NGF-treated PC12 cells (see above and Refs. 13,25,26). Finally, incubation of PC12 cells with PD098059 completely inhibited the NGFstimulated induction of c-Jun protein (Fig. 8B), indicating that c-Jun induction by NGF is ERK-dependent as described previously (29).
The effect of overexpression of c-Jun, JunD, and CREB and different combinations of these transcription factors was tested on the NFLC-luciferase reporter in PC12 cells (Table I). Transfection of c-Jun, JunD, and CREB, but not ATF2, resulted in significant increases in the basal luciferase activity, with c-Jun and CREB stimulating the greatest increases. Moreover, the combination of c-Jun and CREB or JunD and CREB resulted in nearly additive increases in basal promoter activity (Table I).
Co-transfection of c-Jun and JunD or c-Jun, JunD, and CREB did not result in further increases in basal activity relative to c-Jun alone or c-Jun and CREB, respectively. Analysis of the action of transfected c-Jun, JunD, CREB, and ATF2 on NGFstimulated luciferase activity revealed that c-Jun, but not JunD, CREB, or ATF2, enhanced NGF-stimulated activity. Moreover, none of the combinations were significantly different than c-Jun alone. The findings in Table I indicate that c-Jun is limiting for the induction of the NFLC promoter in PC12 cells, whereas CREB and JunD overexpression can significantly increase the basal promoter activity but not the NGF-stimulated activity. To test further the role of c-Jun in regulation of the NFLC promoter in PC12 cells, an expression vector encoding an inhibitory form of c-Jun (c-Jun-DBD) was employed. This construct is predicted to behave in a competitive inhibitory fashion toward endogenous c-Jun (31,32). Compared with basal and NGF-stimulated NFLC promoter activities of 160 Ϯ 28 and 2069 Ϯ 331 RLU/milliunit ␤-Gal, respectively, in control PC12 cells, transfection of c-Jun-DBD yielded basal and NGFstimulated promoter activities of 105 Ϯ 26 and 1136 Ϯ 176 RLU/milliunit ␤-Gal, respectively. Thus, the data in Table I coupled with the ϳ50% decrease in NGF-stimulated NFLC-Luc activity by c-Jun-DBD support the involvement of c-Jun in the regulation of the NFLC promoter by NGF in PC12 cells. DISCUSSION The ability of constitutive active forms of c-Raf-1 and MEK-1 (12,33,34) to induce morphologic differentiation of PC12 cells has led to the suggestion that the ERK pathway is a dominant, perhaps even sufficient, pathway mediating NGF-stimulated  differentiation of PC12 cells (12). However, our findings in this study provide novel evidence for the required collaboration in PC12 cells of the JNK MAP kinase pathway with the ERKs in the regulation of the NFLC gene by NGF. The required integration of Ras and either PLC␥ or c-Src for NGF-stimulated morphological differentiation of PC12 cells has been proposed previously based on findings with mutant Trk and ␤PDGF receptors (7-10). These results are in keeping with our previously published ␤PDGF receptor transfection studies showing a requirement for signaling pathways in addition to the Raf/ MEK/ERK pathway for neurite outgrowth in PC12 cells (9,10). Thus, in contrast to gain-of-function oncogenic forms of c-Raf and MEK1, our findings with NFLC promoter regulation by NGF are similar to those with the egr-1, cyclooxygenase-2, tumor necrosis factor ␣, and atrial natriuretic factor promoters that exhibit a required collaboration of multiple signal pathways for regulation by growth factors and physiologic stimuli (35)(36)(37)(38).
We have observed previously that gene transfer of constitutive active forms of G␣ q induced differentiation of PC12 cells as assessed by neurite extension and Na ϩ channel induction that was correlated with JNK activation (13); ERK activity was not increased in this setting. Likewise, bone morphogenetic protein-2 stimulated morphologic differentiation of PC12 cells, increased NFLC protein content, and was associated with JNK activation but not ERK activation (14). Also, studies with transfected adrenergic receptor sub-types indicate that only the ␣ 1A -adrenergic receptor, which couples to both ERK and JNK MAPKs, is competent to signal PC12 cell differentiation (39). By using conditionally immortalized central nervous system hippocampal neural precursor cells, Rosner and co-workers (40) have established the requirement of Src, Ras, and c-Raf but not ERKs in fibroblast growth factor-induced neural differentiation. Activation of the JNK pathway by fibroblast growth factor was not measured in these studies, but the literature provides ample evidence that both Ras and Src can engage the JNK pathway in different cells (11,41,42). Importantly, the results of our present study move beyond a correlation of JNK activation with PC12 cell differentiation and provide direct evidence for the requirement of both ERK and JNK pathways in the induction of a marker of neural differentiation in PC12 cells.
Our results demonstrate that the NGF-responsive region of the NFLC promoter resides between Ϫ128 and Ϫ38 bp, a region encompassing a CRE/ATF site at Ϫ45 to Ϫ39 bp. Mutation of the CRE/ATF site strongly reduced induction of the NFLC promoter by NGF, indicating that it is necessary for growth factor action (Fig. 6). However, the CRE/ATF site does not appear to be sufficient for full activation since truncation of the NFLC promoter to Ϫ98 bp leaves the CRE/ATF sequence intact but markedly blunts NGF responsiveness of the promoter (Fig. 5). Although data base searches have failed to identify any known consensus enhancer sites between NFLC promoter sequences Ϫ128 and Ϫ98 bp, it is likely that an additional enhancer site or sites resides in this promoter region. The sequence CTGCGTCAG, which includes and flanks the CRE/ATF site in the rat NFLC promoter, is absolutely conserved in sequence and relative positioning to the TATA box in the human and mouse NFLC promoters (43,44), suggesting that this response element functions in an evolutionarily conserved fashion in NFLC gene regulation in vivo. In this regard, several groups have shown that approximately Ϫ300 bp of the 5Ј-flanking sequence of the NFLC gene are sufficient to direct neural specific expression in transgenic mice (44 -46). However, a human NFLC promoter truncated to Ϫ55 bp did not drive neural expression (46), consistent with our finding that sequences proximal to the CRE/ATF site are also required for NGF responsiveness in PC12 cells.
CRE/ATF sites have been invoked as putative regulatory enhancer sites in promoters derived from a number of NGFresponsive genes including nur77, vgf, c-fos, and egr-1 (47)(48)(49)(50)(51). The NGF-responsive nur77 promoter contains two cis-acting elements with a core sequence TGCGTCA identical to the CRE/ ATF sequence in the NFLC promoter, where both are required for induction of the nur77 promoter by NGF (48). In addition, the vgf and c-fos promoters contain the sequence GACGTCA, and the egr-1 promoter contains the sequence, TACGTCA, both of which are highly similar to the NFLC CRE/ATF site. With the exception of the nur77 promoter, where the CRE/ATF sites are sufficient to confer NGF responsiveness to a minimal thymidine kinase promoter (48), the CRE/ATF sites in the vgf, c-fos, and egr-1 promoters are not sufficient to confer NGF inducibility on their own and apparently function in a cooperative manner with other enhancer sites in these promoters. Whereas a CRE/ATF enhancer site may serve an important role in the NGF-dependent regulation of the aforementioned promoters, Sp1 elements appear to serve dominant roles in the control of the p21 WAF1 and cyclin D1 promoters by NGF (28,52). Sp1 sites were not required for NGF-dependent regulation of the NFLC promoter (Fig. 5). Thus, NGF-stimulated transcriptional regulation of the many genes induced during differentiation of PC12 cells likely involves multiple signaling pathways integrating at distinct categories of transcription factor complexes where CRE/ATF sites acting in concert with other cis-acting elements may control a subset of NGF-regulated genes.
Electromobility shift assays identify CREB, JunD, and c-Jun as components of a complex that assembles on the NFLC CRE/ ATF site. CREB is a major factor that binds to the EMSA probe, regardless of whether the nuclear extracts were prepared from control or NGF-stimulated PC12 cells. By contrast, we observed NGF-stimulated recruitment of JunD into the putative regulatory complex that assembles on the CRE/ATF site (Fig.  7B) despite a high constitutive expression of JunD detected by immunoblot analysis (Fig. 8A). JunD has been identified previously as a key factor involved in the regulation of the nur77 gene by NGF in PC12 cells (48). Although the mechanism for JunD recruitment to the CRE/ATF site is not defined in our study, one possibility is heterodimerization with an inducibly expressed member of the bZIP family of transcription factors which is then targeted to this particular enhancer sequence.
Transcriptional activation of the c-jun gene in PC12 cells by NGF occurs in a protein synthesis-independent manner (30) and thus represents an immediate-early response. Evidence also indicates that c-Jun represents a key transcription factor involved in the neural differentiation response elicited by NGF in PC12 cells. We and others (13,29) have reported previously that overexpression of c-Jun in PC12 cells results in morphological differentiation of PC12 cells independent of other upstream signals. Moreover, in the present study, transfection of c-Jun markedly stimulated both the basal and NGF-stimulated activity of the NFLC promoter (Table I), and the c-Jun-DBD construct reduced induction of the NFLC promoter by NGF. Based on the ERK-dependent induction of c-Jun protein by NGF in PC12 cells (Fig. 8 (29)) and the persistent phosphorylation of c-Jun on a JNK-specific site (serine 73) in NGF-treated cells (Fig. 8), we hypothesize that c-Jun serves as a point where the ERK and JNK pathways integrate to regulate the NFLC promoter. This model is consistent with our experiments demonstrating the collaboration of the ERK and JNK pathway at the NFLC promoter shown in Fig. 4, although additional studies will be required to define the precise mechanism by which c-Jun regulates the NFLC promoter. In conclusion, this study suggests that control of gene expression involved in cellular differentiation requires integration of multiple signal pathways and not the action of a single dominant pathway.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Biology
Computer Science

text:
Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

EGF, but not TGFα, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGFα, whereas the ubiquitination was more sustained by incubation with EGF than with TGFα. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGFα, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGFα dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.

Introduction
Several transmembrane proteins are ubiquitinated upon ligation (for reviews see Bonifacino and Weissman, 1998;Hicke, 1999). The EGF receptor (EGFR)* is polyubiquitinated upon activation of the EGFR intrinsic kinase activity (Galcheva-Gargova et al., 1995). c-Cbl was recently demonstrated to possess ubiquitin ligase activity (Joazeiro et al., 1999), and it was further reported that the NH 2 -terminal region of c-Cbl, containing the RING finger responsible for the ubiquitin ligase activity , was sufficient to enhance EGFR downregulation (Lill et al., 2000). c-Cbl has also been demonstrated to negatively regulate PDGF receptor (PDGFR)-dependent cell proliferation by ligand-dependent ubiquitination and subsequent degradation of both PDGFR ␣ and PDGFR ␤ (Miyake et al., 1998(Miyake et al., , 1999. In accordance with these findings, it has been suggested that ubiquitination marks receptors for degradation (Hicke, 1997;Heldin et al., 1998;Levkowitz et al., 1998).
The yeast G protein-coupled receptor Ste2p is accumulated in high molecular weight ubiquitinated forms at the cell surface in response to the binding of ␣ -factor in endocytosis-deficient yeast cells (Hicke and Riezman, 1996). This finding established a link between ligand-induced ubiquitination, endocytosis, and eventual targeting for vacuolar degradation in yeast. It has also been reported that the ubiquitin conjugation system is required for ligand-induced endocytosis of the growth hormone receptor (van Kerkhof et al., 2000). Based on these and other observations, the ubiquitin system has been suggested as an important regulatory system for clathrin-dependent endocytosis (Strous and Govers, 1999). This is in accordance with the demonstration that polyubiquitination of the EGFR occurs at the plasma membrane. We demonstrated that in endocytosis-deficient cells the EGFR was transiently polyubiquitinated, but not degraded (Stang et al., 2000).
It is still unclear to what extent proteasomal processing is involved in downregulation of receptors. In the case of the PDGFR, the degradation was reportedly inhibited Ͻ 20% by proteasome inhibitors (Mori et al., 1995a(Mori et al., , 1995b. The receptor tyrosine kinase c-Met consists of a 50-kD extracellular ␣ subunit and a 140-kD membrane-spanning ␤ subunit. Upon ligand binding, the ␤ subunit was ubiquitinated and subsequently degraded in a proteasome-dependent manner (Jeffers et al., 1997). Also in the case of ErbB2, proteasome inhibitors have been demonstrated to inhibit degradation (Mimnaugh et al., 1996). However, the question of whether or not ErbB2 is in fact endocytosed is still controversial. It was recently reported that the antibiotic geldanamycin induced degradation of ErbB2 and that degradation of the COOH-terminal fragment was prevented by proteasome inhibitors, whereas degradation of the membraneanchored 135-kD fragment was blocked by inhibitors of the endocytosis-dependent degradation pathway (Tikhomirov and Carpenter, 2000). Therefore, the functional role of ubiquitination in endocytosis and downregulation of receptors is unresolved and challenging.
It has been established that EGF and TGF ␣ differ in their ability to induce processing of the EGFR (Ebner and Derynck, 1991). EGF is a more acidic molecule than is TGF ␣ and remains bound to the EGFR upon internalization during acidification of the endocytic vesicle, finally resulting in proteolytic degradation of both ligand and receptor in lysosomes. In contrast, TGF ␣ rapidly dissociates from the receptor upon internalization, resulting in recycling of EGFR to the cell surface (Sorkin and Waters, 1993). We report here that in spite of differential degradation induced by EGF and TGF ␣ , both ligands equally efficiently induce initial ubiquitination and endocytosis of the EGFR. However, EGF, which in contrast to TGF ␣ secures the kinase activity of the EGFR upon endocytosis, maintains a sustained polyubiquitination of the EGFR. Consistently, colocalization of EGFR and c-Cbl on endosomes is observed only upon incubation with EGF. By overexpression of the dominant negative N-Cbl, we confirmed that Cbl-induced ubiquitination is important for degradation of EGF-bound EGFR, but not for endocytosis. By the use of inhibitors of endosomal/lysosomal and proteasomal degradation, we further found that degradation of EGFR and EGF requires proteasomal processing. Therefore, we submit that ubiquitination per se is not a signal for EGFR degradation. However, sustained ubiquitination is required for efficient sorting of the EGFR to a degrading compartment, and proteasomal activity is required for efficient transport of EGF and EGFR to inner membranes of MVBs.

Results
EGFR is efficiently degraded upon incubation with EGF, but not with TGF ␣ To confirm differential degradation of the EGFR upon incubation with EGF compared with TGF ␣ (Ebner and Derynck, 1991), Hep2 cells were incubated with EGF and TGF ␣ in the presence of cycloheximide (CHX), facilitating studies of EGFR degradation by inhibiting protein synthesis. Immunoblotting showed that there was a visible degradation of EGFR already upon incubation with EGF for 2 h ( Fig. 1 A, top). In contrast, there was only a slight degradation of the EGFR upon incubation with TGF ␣ for as long as 7 h ( Fig. 1 A, bottom). pH decreases gradually in endocytic vesicles. Whereas intravesicular pH is ف 6.0 in early endosomes, the pH of lysosomes is 4.5-5.0 (Yamashiro and Maxfield, 1984). The EGFR is fully phosphorylated on ice (unpublished data). However,the phosphorylation is pH sensitive (Fig. 1 B). In cells incubated with TGF ␣ , the EGFR became dephosphorylated upon exposure to slightly reduced pH. The EGFR in cells incubated with EGF was dephosphorylated only at pH values similar to those found in lysosomes. These results can be explained by the fact that TGF ␣ is released from the EGFR at slightly reduced pH values, whereas EGF is not. Accordingly, TGF ␣ will be released in early endosomes, allowing the EGFR to recycle to Figure 1. Degradation and pH dependent phosphorylation of the EGFR upon incubation with EGF and TGF␣. (A) Hep2 cells were incubated with CHX (25 g/ml) and EGF (10 nM) (top) or TGF␣ (10 nM) (bottom) at 37ЊC for the indicated time periods. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR (Stang et al., 2000). c, control cells not incubated with ligand nor CHX. (B) Hep2 cells were incubated with EGF (10 nM) or TGF␣ (10 nM) on ice for 15 min. The medium was removed, and the cells were incubated on ice for 20 min in MEM without bicarbonate with BSA and MES (10 mM) adjusted to pH 5.0, 6.0, 7.0 and 7.4, containing EGF (10 nM) or TGF␣ (10 nM). The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to activated EGFR (pY1173). (C) Hep2 cells were incubated with 10 nM EGF (᭡) or 10 nM TGF␣ (᭝) at 37ЊC for indicated times, before the cells were biotinylated on ice. Biotinylated EGFR was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting. Biotinylated EGFR was normalized for precipitation efficiency using total EGFR immunoprecipitated. The data represent the mean of four independent experiments Ϯ SEM. the plasma membrane. However, EGF will remain complexed to the EGFR securing EGFR kinase activity and thereby lysosomal sorting. The efficiency of endocytosis induced by EGF and TGF ␣ was assessed by measuring the amount of surface-localized (noninternalized) EGFR by biotinylation. As demonstrated in Fig. 1 C, and as previously reported (Decker, 1990;Ebner and Derynck, 1991), EGF and TGF ␣ induced endocytosis with the same efficiency.
The difference in intracellular trafficking of the EGFR upon activation with EGF versus TGF ␣ , was further studied using immuno-EM. Labeling of thawed cryosections showed that the early events in endocytosis were identical upon stimulation with EGF and TGF ␣ . Upon binding of ligand to cells on ice and subsequent chase at 37 Њ C, the EGFR was internalized via clathrin coated pits. After a 10min chase, the labeling was found in compartments with tubulovesicular morphology typical of early endosomes (unpublished data). However, after a 30-min chase, the intracellular labeling pattern and intensity showed liganddependent differences. Whereas with both ligands labeling was found associated with MVBs ( Fig. 2), the morphology of the EGFR-positive MVBs as well as the localization of the EGFR within MVBs varied. In cells incubated with TGF ␣ , labeling intensity was low and mainly found on MVBs containing only a few internal vesicles (Fig. 2, D-E). This possibly represents MVBs at an early stage of formation, and the labeling was largely restricted to the outer, limiting membrane of the MVBs. However, in cells incubated with EGF, labeling was found on MVBs at all stages of formation, and labeling for both EGF and the EGFR was concentrated on the internal vesicles (Fig. 2, A-C). Quantification of EGFR labeling showed that whereas in cells incubated with TGF ␣ only 25% of the labeling was localized to inner membranes, Ͼ 80% showed this localization upon incubation with EGF. A similar difference in trafficking has previously been shown between kinase active and kinase inactive EGFR (Felder et al., 1990), demonstrating that the EGFR kinase activity is needed for sorting of the EGFR to internal vesicles within MVBs. In addition to labeling on MVBs, some EGFR was found within small vesicles close to the MVBs. The nature of these vesicles is unknown. However, as this labeling appeared to be most frequent in cells incubated with TGF ␣ , the vesicles might be involved in recycling of the EGFR. Recycling is probably also the reason why intracellular labeling intensity was low upon 30 min chase upon incubation of cells with TGF ␣ . The different labeling found in our experiments probably reflects the different physiological character of EGF and TGF ␣ as EGFR ligands. EGF remains bound to the EGFR, keeping the kinase active within MVBs, whereas TGF ␣ is dissociated in MVBs, and the EGFR kinase is no longer active ( Fig. 1 B).
Both EGF and TGF ␣ induce polyubiquitination of EGFR, but the ubiquitination is more sustained with EGF than with TGF ␣ Ubiquitination of the EGFR was studied by immunoprecipitating conjugated ubiquitin and immunoblotting the immunoprecipitated material using an antibody to EGFR. As demonstrated in Fig. 3 A, both EGF and TGF ␣ induced ubiquitination of the EGFR. However, the amount and af- Figure 2. The EGFR shows different localization within MVBs upon incubation with EGF compared with TGF␣. Hep2 cells incubated with EGF (10 nM) (A-C) or TGF␣ (10 nM) (D-E) on ice for 15 min and chased at 37ЊC for 30 min were processed for immuno-EM, sectioned and labeled using antibody to EGF (A) or EGFR (B-E). Upon activation with EGF, labeling for EGF (A), as well as for EGFR (B-C), is found on MVBs at all stages of formation. The labeling on MVBs is found on the limiting membrane (small arrowheads), as well as on internal vesicles. In MVBs with a high number of internal vesicles (B and C), labeling is concentrated on the inner membranes. Some labeling for the EGFR is also found on small vesicles within the cytoplasm (large arrowheads). In cells activated with TGF␣ (D-E), labeling intensity is generally reduced (D), and labeling is mainly localized to the outer limiting membrane of MVBs. Note that the labeling of small vesicles within the cytoplasm appears to be most frequent in TGF␣-treated cells. Bar, 100 nm. finity of the antibody used could potentially be a limiting factor with respect to immunoprecipitation of the protein of interest. To exclude the possibility that EGF induces a stronger initial ubiquitination than does TGF ␣ , cell lysates were subjected to SDS-PAGE and immunoblotted with antibody to EGFR. By overexposing the film, we clearly observed polyubiquitination of the EGFR as "upsmearing." As demonstrated in Fig. 3 A, top and middle, both EGF and TGF ␣ induced the same initial ubiquitination of EGFR. However, the ubiquitination induced by EGF was more sustained than the ubiquitination induced by TGF ␣ . EGF also induced a more sustained phosphorylation of EGFR than did TGF ␣ (Fig. 3 A, bottom). Interestingly, ubiquitination of the EGFR occurred even at 0 Њ C, before endocytosis of EGFR ( Fig. 3 A, top).
c-Cbl is the ubiquitin ligase responsible for the EGFmediated ubiquitination of the EGFR Yokouchi et al., 1999). Therefore, we investigated activation of c-Cbl in Hep2 cells upon EGF-and TGF ␣ -induced activation of EGFR. Immunoblotting of c-Cbl immunoprecipitates with antibody to phosphotyrosine showed that c-Cbl was tyrosine phosphorylated upon incubation both with EGF and with TGF ␣ on ice for 15 min and remained phosphorylated during chase at 37 Њ C for 30-60 min ( Fig. 3 B). To confirm that the EGF/TGF ␣ -induced ubiquitination of the EGFR is dependent upon EGFR kinase activity, Hep2 cells were preincubated with the EGFR kinase inhibitor PD153035 for 2 h at 37 Њ C, before incubation with EGF. PD153035 efficiently inhibited ubiquitination of the EGFR (

TGF ␣ and EGF recruit c-Cbl to the plasma membrane, whereas only EGF recruits c-Cbl to endosomes
Immunocytochemical labeling for confocal microscopy was performed, using antibodies to EGFR and c-Cbl. The specificity of the antibodies was investigated by Western blotting (unpublished data) and further confirmed by ligand-induced changes in localization of EGFR and c-Cbl. The EGFR was mainly localized to the plasma membrane in control cells (Fig. 4 A) and in cells incubated with EGF or TGF ␣ on ice (Fig. 4, D and G). Whereas c-Cbl in control cells mainly localized to the cytoplasm ( Fig. 4 B), c-Cbl was recruited to the plasma membrane upon incubation both with EGF and with TGF ␣ on ice (Fig. 4, E and H) and appeared to colocalize with the EGFR (Fig. 4, F and I). In cells incubated with EGF, both the EGFR and c-Cbl were redistributed and colocalized on vesicular structures upon chase at 37 Њ C for 15 min (Fig. 4, J-L). Upon binding of TGF ␣ and further chase at 37 Њ C for 15 min, the EGFR localized both to the plasma membrane and to vesicular structures ( Fig. 4 M). However, c-Cbl, relocalized to the cytoplasm ( Fig. 4 N), and there was no clear colocalization of EGFR and c-Cbl (Fig. 4 O).

Dominant negative Cbl (N-Cbl) inhibits degradation, but not endocytosis of EGF and EGFR
Ubiquitination of the EGFR at the plasma membrane could be important for endocytosis of the EGFR. To address this, we transiently transfected COS-1 cells with a plasmid en- The cells were lysed, and the lysates were subjected to immunoprecipitation with antibody to conjugated ubiquitin, followed by immunoblotting with antibody to EGFR (top). Lysates were also subjected to SDS-PAGE and immunoblotting with antibody to EGFR (middle) or activated EGFR (pY1173) (bottom). c, control cells not incubated with ligand. (B) Hep2 cells were incubated as in (A), and c-Cbl was immunoprecipitated. The precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to phosphotyrosine (pTyr). c, control cells not incubated with ligand. (C) Hep2 cells were preincubated with PD153035 (100 nM) for 2 h at 37ЊC, before incubation with EGF (10 nM) on ice for 15 min. The cells were subsequently chased in MEM without bicarbonate containing PD153035 (100 nM) at 37ЊC for the indicated time periods. Conjugated ubiquitin was immunoprecipitated, and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to EGFR (left). c-Cbl was immunoprecipitated, and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibody to phosphotyrosine (pTyr) (right).
coding HA-tagged N-Cbl. N-Cbl consists of the NH 2 -terminal 357 amino acids of c-Cbl, comprising the SH2 domain, but not the RING finger domain responsible for EGFR ubiquitination. When overexpressed, N-Cbl will bind to phosphorylated Tyr 1045 of the EGFR and inhibit binding of c-Cbl, thereby inhibiting EGFR ubiquitination. Transfected and nontransfected cells were compared with respect to internalization of Rh-EGF. As demonstrated in Fig. 5 A, cells overexpressing N-Cbl (identified with anti-body to hemagglutinin [HA]) internalized Rh-EGF as efficiently as did nontransfected cells. The rate of internalization and degradation of EGF was further studied in COS-1 cells transfected with the plasmid encoding HA-tagged N-Cbl. As demonstrated in Fig. 5 B, the internalization of 125 I-EGF (0.2 nM) was not altered upon transfection with the N-Cbl plasmid. Degradation and recycling was investigated upon loading transfected cells with 125 I-EGF (8.5 nM). Degradation was clearly inhibited, and recycling was in- The cells were processed and immunostained, as described in Materials and methods, using sheep anti-EGFR (1:500) and rabbit anti-c-Cbl (1:500), followed by Cy™2-conjugated donkey anti-sheep (1:250) (green) and Alexa Fluor 594conjugated goat anti-rabbit (1:1,000) (red). In nonstimulated cells the EGFR is concentrated at the plasma membrane (A, arrowhead), whereas c-Cbl is localized to the cytoplasm. Upon incubation with EGF or TGF␣ on ice, c-Cbl is recruited to the plasma membrane and colocalizes with EGFR (D-I, arrowheads). The EGFR colocalizes with c-Cbl in vesicular compartments upon incubation with EGF and chase at 37ЊC for 15 min, as indicated by arrowheads in J-L. After incubation with TGF␣ and chase at 37ЊC for 15 min the EGFR is localized at the plasma membrane, and c-Cbl is localized to the cytoplasm, giving no colocalization. Bar, 50 m. creased (Fig. 5 C). The degradation (Fig. 5 D) as well as the ubiquitination (Fig. 5 E) of the EGFR was inhibited in N-Cbl-transfected cells. The data presented are an underestimate of the actual effects of overexpression of N-Cbl, as the transfection efficiency was %02ف (estimated as the percentage of cells reactive to anti-HA antibody). The data reflected degradation/recycling/ubiquitination in the total cell population. Together, the data support the notion that ubiquitination of the EGFR does not promote internalization, but is instrumental in sorting the EGFR from early to late endosomes.

NH 4 Cl, MG132 and lactacystin inhibit EGF-mediated degradation of the EGFR
The differential effect of EGF and TGF␣ with respect to degradation of the EGFR cannot be explained by differences in initial ubiquitination, nor by differences in endocytosis. However, only EGF caused a sustained ubiquitination, probably induced upon endocytosis. Degradation of the liganded EGFR is known to occur in lysosomes (Carpenter and Cohen, 1976;King et al., 1980aKing et al., , 1980b. However, in analogy with other ubiquitinated receptors, proteasomes could additionally be involved in the degradation of the EGFR. To study this possibility, we investigated the effect of NH 4 Cl, which counteracts the acidification of endocytic vesicles, and of the proteasomal inhibitors MG132 and lactacystin, on EGFR degradation. Hep2 cells were incubated with EGF (10 nM) in the presence of CHX and in the presence or absence of NH 4 Cl, MG132 or lactacystin. As demonstrated in Fig. 6 A, NH 4 Cl, MG132, and lactacystin all inhibited degradation of the EGFR, and MG132 was the most potent inhibitor of degradation. The observed EGFR degradation was the same when an antibody raised against the extracellular part of the EGFR was used (unpublished data). The data strongly suggest that lysosomal degradation of EGFR depends on proteasomal activity. We also investigated the effect of the same inhibitors with respect to EGFR activation by immunoblotting with an antibody to pY1173. As demonstrated in Fig. 6 B, phosphorylation of EGFR was sustained in the presence of NH 4 Cl, MG132 and lactacystin, and MG132 affected the phosphorylation most efficiently. The sustained phosphorylation most likely reflects inhibited degradation of the phosphorylated receptor.
To exclude the possibility that the inhibition of EGFR degradation was a result of inhibited endocytosis, internalization of 0.2 nM 125 I-EGF was investigated in the presence or absence of NH 4 Cl, MG132, or lactacystin. Hep2 cells EGF (10 nM) was added to mock-transfected COS-1 cells (control) and to N-Cbl-transfected COS-1 cells, and EGF was bound for 15 min on ice. Then the cells were washed, and subsequently incubated at 37ЊC for 10 min. The cells were lysed and the lysate immunoprecipitated with antibody to conjugated ubiquitin. Immunoprecipitated material was subsequently analyzed by SDS-PAGE and immunoblotting with anti-EGFR antibody.
were preincubated with NH 4 Cl, MG132 or lactacystin at 37ЊC before incubation with 125 I-EGF on ice for 15 min. The cells were subsequently washed and incubated at 37ЊC with medium containing the inhibitors. As shown in Fig. 6 C, neither NH 4 Cl, MG132, nor lactacystin, had any effect on the internalization of 125 I-EGF. Both NH 4 Cl and MG132, but not lactacystin, inhibited degradation of 125 I-EGF at the low concentration of ligand used in this experi- Figure 6. Degradation of the EGFR and 125 I-EGF in the presence of NH 4 Cl, MG132, and lactacystin. (A-C) Hep2 cells were preincubated with either NH 4 Cl, MG132 or lactacystin, as described in Materials and methods. (A) EGF (10 nM) and CHX (25 g/ml) was added, and the cells were further incubated at 37ЊC for the indicated time periods. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR. c, control cells not incubated with ligand or CHX. Both NH 4 Cl, MG132 and lactacystin inhibited the degradation of EGFR. The presented data is representative of seven independent experiments. (B) EGF (10 nM) was added to cells on ice for 15 min before chase at 37ЊC for the indicated times in medium containing the same inhibitors as during the preincubation. The cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to activated EGFR (pY1173). c, control cells not incubated with ligand. (C) Cells preincubated with or without inhibitors were incubated with 125 I-EGF (0.2 nM) on ice for 15 min and chased in medium containing the inhibitors at 37ЊC for the indicated time periods. Analysis of EGF in the medium (᭞), EGF at the cell surface (᭺), internalized EGF (᭹) and degraded EGF (᭢) was performed as described (Skarpen et al., 1998). Both NH 4 Cl and MG132, but not lactacystin, inhibited the degradation of 125 I-EGF. (D) Hep2 cells were preincubated for 2 h with and without lactacystin. The cells were then loaded with 8.5 nM 125 I-EGF for 20 min at 37ЊC, and recycling and degradation was investigated as described in Fig. 5 C. Lactacystin was found to enhance recycling and to inhibit degradation of 125 I-EGF. The fraction representing intact intracellular EGF was not affected by lactacystin and is not demonstrated. The data in C and D represent the mean of 3 independent experiments Ϯ SEM. ment. To study effects of lactacystin on recycling and degradation of EGFR-bound EGF, 8.5 nM 125 I-EGF was used, and the analysis was performed essentially as described (Babst et al., 2000). As demonstrated in Fig. 6 D, lactacystin slightly enhanced recycling and inhibited degradation of 125 I-EGF. The fact that lactacystin inhibits degradation of EGFR-bound EGF at high ligand concentration suggests that proteasomal activity is involved in efficient delivery to an environment where EGF is degraded.
The finding that MG132, but not lactacystin, inhibits degradation of very low concentrations of EGF can be explained by the possibility that MG132, which is a peptide aldehyde, does not specifically inhibit proteasomal proteases. We used a Cathepsin B enzyme assay to study the potential effect of MG132 and lactacystin on Cathepsin B, reported to be important for degradation of EGF and EGFR (Authier et al., 1999). We found that MG132 caused an 80% inhibition of Cathepsin B, whereas lactacystin only slightly (Ͻ10%) inhibited Cathepsin B. Therefore, the strong inhibitory effect of MG132 on degradation of EGF can be explained by the inhibitory effect of MG132 on Cathepsin B.
The effect of lactacystin on ligand-induced trafficking of the EGFR was further studied by immuno-EM. Control cells or cells pretreated with lactacystin or MG132 were incubated with EGF (10 nM) for 15 min on ice, followed by chase in ligand-free, prewarmed medium with inhibitor. After 1 h chase, both EGF and the EGFR (unpublished data) localized to MVBs. However, both the appearance of MVBs and the distribution of EGF and EGFR inside MVBs were different in control cells compared with in cells treated with lactacystin and MG132. In control cells the labeling for EGF was found in MVBs with a high number of internal vesicles, and Ͼ80% of the labeling localized to these internal membranes (Fig. 7 A). In cells treated with lactacystin or MG132 (Fig. 7 B; unpublished data), labeling for EGF was mainly found on MVBs with few internal vesicles, and Ͼ60% of the labeling was on the outer limiting membrane. This shows that proteasomal activity is involved in the translocation of EGFR from the limiting membrane to internal membranes within MVBs.
To investigate whether proteasomal inhibitors affected all intracellular sorting, we studied the fluid phase endocytosis of BSA-coated colloidal gold. As demonstrated in Fig. 7 C, BSA-gold was transported efficiently to MVBs filled with internal membranes, demonstrating that lactacystin did not affect formation of MVBs in general.
To characterize the endosomes to which EGF and EGFR localize in the presence of lactacystin more closely, we added Rh-EGF and FITC-transferrin (Tf) to cells in the presence or absence of lactacystin. Upon incubation for 15 min, most EGF colocalized with Tf. Upon incubation for 60 min, there was no significantly increased colocalization of EGF and Tf in lactacystin-treated cells, compared with control cells (unpublished data). Therefore, the endosomes depicted in Fig. 7 B do not appear to be part of the recycling compartment.

Proteasomal inhibitors do not inhibit degradation of EGF-EGFR by depleting cellular ubiquitin
A side effect of incubating cells with proteasomal inhibitors could be depletion of intracellular ubiquitin (Swaminathan et al., 1999). Therefore, we investigated the effect on EGFR ubiquitination of incubating Hep2 cells with lactacystin for 3 and 5 h. Hep2 cells were preincubated with both lactacystin and CHX for 3 and 5 h (the same way degradation of EGFR was studied). As shown in Fig. 8, the EGFR was ubiquitinated equally efficiently by addition of EGF regardless of preincubation with lactacystin. This demonstrates Figure 7. Translocation of EGF-EGFR to internal membranes of MVBs depends on proteasomal activity. Control and lactacystintreated Hep2 cells were incubated with EGF for 15 min on ice followed by chase in EGF free medium at 37ЊC for 1 h. To localize endocytosed EGF, ultra-thin frozen sections were labeled with anti-EGF antibodies followed by 15 nm protein A gold. In control cells (A) EGF is localized within MVBs with a high number of internal vesicles, and small amounts of labeling is also found within electron dense, compact MVBs (arrowhead). In lactacystin-treated cells (B) EGF is localized to MVBs with very few internal vesicles. (C) Lactacystin-treated Hep2 cells (preincubated for 2 h) were incubated with 10 nm BSA-gold for 60 min at 37ЊC. Endocytosed gold was found to localize within MVBs with high numbers of internal vesicles. Bars, 100 nm.

Figure 8. Preincubation of Hep2 cells with lactacystin for 3 and 5 h does not inhibit subsequent EGF-induced ubiquitination of the EGFR.
Hep2 cells were preincubated with or without lactacystin and CHX for 3 or 5 h. Then control cells and cells pretreated with lactacystin/CHX were incubated without or with EGF for 15 min on ice before being washed and incubated at 37ЊC for 10 min. The cell lysates were subjected to SDS-PAGE and immunoblotting with antibody to EGFR. The upsmearing illustrates ubiquitination. that reduction of intracellular ubiquitin resulting from preincubation of cells with lactacystin was not sufficient to inhibit EGF-induced polyubiquitination of the EGFR and strongly supports the notion that proteasomal activity is essential for efficient transport of EGF-bound EGFR to inner membranes of MVBs.

The EGFR is not a direct target of proteasomal proteases
An important consideration is the nature of the proteasomal substrate. To clarify whether the EGFR is itself cleaved during sorting to MVBs, we transfected Hep2 cells with a plasmid encoding the EGFR-green fluorescent protein (GFP) fusion protein. This fusion protein is endocytosed and degraded exactly like wild-type EGFR upon binding EGF (Carter and Sorkin, 1998). Therefore, we investigated by immuno-EM whether or not GFP could be detected on inner membranes of MVBs. As illustrated in Fig. 9, we found that upon incubation of the cells with EGF for 30 min at 37ЊC, labeling for the EGFR as well as for GFP could be detected both on the limiting and internal membranes of MVBs. Quantification showed that %06ف of anti-EGFR labeling and 50% of the anti-GFP labeling localized to the internal membranes. As the anti-EGFR antibody recognizes the cytosolic part of the EGFR and GFP is a COOH-terminal extension of the EGFR, the experiment demonstrates that the cytosolic part of the EGFR is intact even when the EGFR localizes to internal membranes of MVBs. This shows that although proteasomal activity is needed for efficient translocation of EGFR to internal membranes of MVBs, the EGFR itself is not a direct proteasomal target.

Discussion
In the present work we have expanded on our previous observation that the EGFR was polyubiquitinated at the plasma membrane (Stang et al., 2000). We have studied EGFR ubiquitination using Hep2 cells, and we now report that the EGFR becomes polyubiquitinated even when cells are incubated with ligand on ice. As endocytosis of the EGFR does not happen when cells are incubated on ice, our results confirm the finding that EGFR is ubiquitinated at the plasma membrane before endocytosis. Consistently, we observed tyrosine phosphorylation of c-Cbl and recruitment of c-Cbl to the plasma membrane upon activation of the EGFR on ice. A possible scenario is that c-Cbl-dependent ubiquitination is important for ligand-induced endocytosis of the EGFR. This would be in agreement with the conclusion that c-Cbl is required for endocytosis of the colony stimulating factor-1 receptor (Wang et al., 1996Lee et al., 1999). However, our present results demonstrate that overexpression of N-Cbl, which acts dominant negatively with respect to ubiquitination of the EGFR, does not affect EGFR endocytosis. This is in accordance with previously published views (Levkowitz et al., 1998;Thien et al., 2001).
We have taken advantage of the two different EGFR ligands, EGF and TGF␣, to induce different trafficking of the EGFR upon endocytosis. Interestingly, both ligands initially induced ubiquitination and endocytosis of the EGFR to the same extent, even though TGF␣ very inefficiently induced transport of the EGFR to internal vesicles of MVBs and lysosomal degradation of the EGFR. This clearly illustrates that the initial ubiquitination induced by activation of the EGFR is not a signal for lysosomal sorting. It should be noted that the ubiquitination induced by EGF lasted longer than the ubiquitination induced by TGF␣. The sustained ubiquitination induced by EGF correlated with the sustained tyrosine phosphorylation of the EGFR and c-Cbl, and therefore the sustained kinase activity of the EGFR. Because EGFR kinase activity has been demonstrated to be required for transport of the EGFR from the limiting membrane to the internal vesicles of MVBs (Felder et al., 1990), the sustained ubiquitination seen upon incubation with EGF could be important for trafficking of EGFR to lysosomes. Interestingly, Katzmann et al. (2001) recently reported ubiquitin-dependent sorting of the vacuolar hydrolase carboxypeptidase S to internal membranes of MVBs in yeast. They further characterized a conserved endosomal sorting complex containing the protein Vps23. This protein contains a ubiquitin conjugating-like domain (Babst et al., 2000). The mammalian homologue of Vps23 is the protein encoded by the tumor susceptibility gene 101 (Tsg101), and this protein has been demonstrated to function in late endosomal trafficking. In fact, in tsg 101 mutant cells, endocytosed EGFR was rapidly recycled back to the cell surface and inefficiently degraded (Babst et al., 2000). Katzmann et al. proposes a model whereby ubiquitinated proteins bind to the sorting complex containing Vps23. Several other class E Vps proteins are suggested to be important for the subsequent sorting, as well as for recruitment of deubiquitinating enzymes, like Doa4 (Katzmann et al., 2001).
The importance of proteasomal activity in lysosomal trafficking of EGFR is supported by our finding that lactacystin inhibits degradation of the EGFR as efficiently as does NH 4 Cl. Our present immuno-EM data show that inhibition of proteasomal activity with lactacystin causes retention of EGFR in MVBs at an early stage of formation. Consistently, Figure 9. The COOH-terminal part of EGFR-GFP is intact upon translocation of EGFR to inner membranes of MVBs. Hep2 cells transfected with EGFR-GFP, as described in Materials and methods, were incubated with 10 nM EGF for 30 min at 37ЊC before processing for immuno-EM. Thawed cryosections were double labeled using anti-GFP antibodies followed by 15 nm protein A gold and anti-EGFR antibodies followed by 10 nm protein A gold (arrowheads). The results show that both GFP-and EGFR-labeling localize to the limiting membrane as well as to inner membranes of MVBs. Bar, 100 nm.
we found, as did Levkowitz et al. (1998), that inhibition of proteasomes promoted recycling and inhibited degradation of EGF. However, the effect of lactacystin on recycling and degradation of EGF was smaller than the effect observed in tsg 101 mutant cells (Babst et al., 2000) and smaller than the effect observed upon overexpression of N-Cbl (transfection efficiency considered). That degradation of EGF is inhibited more efficiently by overexpression of N-Cbl and in tsg 101 mutant cells compared with in cells incubated with lactacystin, suggests the involvement of proteasomes downstream of ubiquitinated EGFR interacting with a complex containing Tsg 101. Our data show that proteasomal activity is not required for the formation of MVBs in general, as lactacystin does not inhibit transport of BSA-gold to MVBs. Morphologically the EGF/EGFR positive compartments found upon inhibition of proteasomal activity resembled the EGFR positive compartments found upon incubation with TGF␣. We found no significantly increased colocalization of EGF and Tf upon incubating cells with lactacystin.
Although ubiquitinated EGFR can be recognized and degraded by purified 26S proteasomes in vitro , our results show that the cytosolic part of the EGFR is intact on internal membranes of MVBs, and therefore demonstrate that the EGFR itself is not a direct proteasomal target in vivo. It was earlier also demonstrated that although the ubiquitination machinery and proteasomal activity is needed for degradation of the growth hormone receptor, neither the cytosolic tail of the growth hormone receptor is a proteasomal target (van Kerkhof et al., 2001). Therefore, the nature of the proteasomal target(s) is still unclear.
In conclusion, our data demonstrate that there is an initial ubiquitination of the EGFR at the plasma membrane, but that this ubiquitination per se does not encode EGFR degradation and that initiation of endocytosis of the EGFR does not depend on ubiquitination. Upon conditions where the EGFR kinase activity is secured, the ubiquitination of the EGFR is sustained. Eventually, activated EGFR is efficiently transported to internal vesicles of MVBs in a proteasome dependent process. Therefore, the sustained ubiquitination of the EGFR seems to be a way of engaging ubiquitin-interacting proteins, and proteasomal cleavage eventually facilitates the transport of the EGFR to a highly degrading environment.

Materials
Human recombinant EGF was from Bachem Feinchemikalien AG, human recombinant TGF␣ was from R&D Systems, Inc., 125 I-EGF was from Amersham Pharmacia Biotech, MG132 from Affiniti Research Products Ltd., and PD153035 was from Tocris Cookson Ltd. EZ-link sulfo-NHS-LC-Biotin was from Perbio Science Ltd., alkaline phosphatase-conjugated streptavidine from Dako Corporation, and rhodamin-EGF (Rh-EGF) and FITC-Tf were from Molecular Probes, Inc. BSA-gold was prepared according to Slot and Geuze (Slot and Geuze, 1985). Fugene was from Roche Diagnostics Corporation. Other chemicals were from Sigma-Aldrich unless otherwise noted.

Cell culture and treatment
Hep2 and COS-1 cells were grown in DME (BioWhittaker) containing penicillin-streptomycin-fungizone mixture (BioWhittaker), L-glutamine (2 mM) (BioWhittaker), and FBS (BioWhittaker) (5% vol/vol in the case of Hep2 cells and 10% for COS-1 cells). The cells were plated at a density of 15,000 cells/cm 2 48 h before experiments. In pulse-chase experiments, cells were incubated with ligand in MEM without bicarbonate with 0.1% BSA on ice for 15 min, followed by washing three times in ice-cold PBS to remove unbound ligand, and subsequent chase in ligand-free MEM without bicarbonate at 37ЊC.

Antibodies
Sheep anti-EGFR was from Gibco Life Technologies, and Fitzgerald Industries International, Inc., rabbit anti-EGFR, rabbit anti-EGF, and rabbit antic-Cbl from Santa Cruz Biotechnology, Inc., mouse antiactivated EGFR (pY1173) and mouse anti-phosphotyrosine from Upstate Biotechnology, rabbit anticonjugated ubiquitin from Sigma-Aldrich, mouse anti-hemagglutinin (HA) from Roche Diagnostics, rabbit anti-GFP from AbCam Ltd., rabbit anti-mouse IgG from Cappel, ICN Biomedicals. Peroxidase-conjugated donkey anti-sheep, peroxidase-conjugated donkey anti-mouse, Cy TM 2-conjugated donkey anti-sheep and rabbit anti-sheep IgG were all from Jackson ImmunoResearch Laboratories. Alexa Fluor 594-conjugated goat anti-rabbit was from Molecular Probes.

Immunoprecipitation
Cells were lysed in immunoprecipitation buffer, as described (Stang et al., 2000). Protein A or protein G-coupled Sepharose beads (Amersham Pharmacia Biotech) were incubated with antibody for 1 h at room temperature and subsequently washed twice with immunoprecipitation buffer, before the cell lysates were added. Immunoprecipitation was performed at 4ЊC for 1 h. Immunoprecipitation with antibody to conjugated ubiquitin and SDS-PAGE and immunoblotting was performed as described (Stang et al., 2000).

Biotinylation of surface-localized EGFR
Biotinylation was performed at 4ЊC, essentially as described (Corbeil et al., 1999), using 2 mM biotin. The EGFR was immunoprecipitated using rabbit anti-EGFR and protein A-coupled Sepharose beads (Amersham Pharmacia Biotech). The immunoprecipitate was subjected to SDS-PAGE and electrotransferred to PVDF transfer membrane (Hybond-P; Amersham Pharmacia Biotech). The membrane was incubated with alkaline phosphataseconjugated streptavidin or with sheep anti-EGFR antibody and alkaline phosphatase-conjugated anti-sheep antibody. Reactive bands were analyzed by enhanced chemifluoresence (Amersham Pharmacia Biotech). The band intensity was measured using a phosphorofluoroImager (Molecular Imager FX; Bio-Rad Laboratories).

Plasmids and transfection of cells
The plasmid EGFR-GFP, encoding a fusion protein of EGFR and enhanced GFP (Carter and Sorkin, 1998) was provided by Dr. Alexander Sorkin. Hep2 cells were transfected with this plasmid using Fugene 24 h upon plating. HA-tagged c-Cbl in the pJZenNeo vector (Andoniou et al., 1994) was provided by Dr. Robin M. Scaife. By polymerase chain reaction a 1,121-bp fragment, corresponding to the HA tag and the 357 amino acids constituting N-Cbl was subcloned into pCDNA3. This plasmid was transfected into COS-1 cells using Fugene 24 h after plating. Transfected cells were analyzed 48 h after transfection.

Immunocytochemistry and confocal microscopy
Cells were plated on 12-mm coverslips (MENZEL-GLÄSER ® ). After experiments, cells were washed once in cytoskeleton buffer ( mM]) and subsequently fixed with paraformaldehyde (4% wt/vol) (Riedel-de Haën AG) in Soerensen's phosphate buffer for 20 min on ice. Cells were washed three times with cytoskeleton buffer before permeabilization with Triton X-100 (0.1% wt/vol in cytoskeleton buffer) for 10 min. The fixed and permeabilized cells were preincubated with BSA (1% wt/vol in PBS) for 30 min before incubation with primary antibodies for 1 h. The coverslips were washed with PBS and incubated with secondary antibodies for 30 min before mounting with Dako fluorescent mounting medium with NaN 3 (15 mM). The cells were examined using a confocal microscope (Leica TCS).

I-EGF interaction experiments
The cells were incubated as described in legends to Figs. 5 and 6. Internalization and degradation of EGF was analyzed as previously described (Skarpen et al., 1998). Additionally, recycling of EGF was analyzed essentially as described (Babst et al., 2000), loading cells with 8.5 nM 125 I-EGF in MEM without bicarbonate and with 0.1% BSA. Upon loading, the surface-localized radioactivity was removed by a glycine-buffered solution, pH 3.0 (Babst et al., 2000). Upon chase at 37ЊC, the medium was analyzed for degraded and recycled EGF as described (Skarpen et al., 1998), and the cells were analyzed for recycled EGF (released by the pH 3.0 buffer) and for internalized EGF (cells solubilized by 1 M NaOH after treatment with low pH). The cpm representing recycled EGF in the medium and at the cell surface were combined in one fraction.

Inhibition of lysosomal or proteasomal activity
Hep2 cells were preincubated with either NH 4 Cl (10 mM) for 10 min, MG132 (10 M) or lactacystin (50 M) for 1 or 2 h at 37ЊC. To measure degradation of the EGFR, EGF (10 nM) and CHX (25 g/ml) were added and the cells incubated at 37ЊC for the indicated times. Cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR. For immuno-EM, cells preincubated with the different inhibitors were incubated with EGF (10 nM) on ice for 15 min followed by chase at 37ЊC for the indicated time periods in the presence of inhibitors.

Cathepsin B enzyme assay
The activity of Cathepsin B (Calbiochem) in the presence of lactacystin and MG132 was analyzed by use of Colorimetric Substrate I (Calbiochem) according to the manufacturer's instructions. The enzyme assay was performed three times with three parallels for each condition.

Immuno-EM
The cells were incubated as described in legends to figures. The cells were subsequently washed with PBS, fixed with paraformaldehyde (4% wt/vol) and glutaraldehyde (0.1% wt/vol) in Sorensen's phosphate buffer and processed for cryosectioning and immunolabeling (Griffiths et al., 1984). Bound antibodies were visualized using protein A gold, a gift of Dr. G. Posthuma. When the primary antibody was mouse or sheep IgG, incubation with rabbit anti-mouse or rabbit anti-sheep IgG was used as an intermediate step between the primary antibody and protein A gold. The sections were examined using a Philips CM 120 electron microscope. To quantify labeling of MVBs, the total number of gold particles found on MVBs (100%) were divided into two groups, gold particles associated with the outer limiting membrane and gold particles associated with internal membranes, and the percentage distribution of the labeling was calculated. In each experiment a minimum of 100 gold particles were counted on MVBs.