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The E5 protein of human papillomavirus type 16 (HPV16) is a small hydrophobic protein, which localizes to the cell membrane, Golgi apparatus and endosomes. HPV16 E5 enhances the activation of the epidermal growth factor (EGFR). The activated EGFR is downregulated through the endocytic pathway, where E5 has been shown to inhibit endosomal acidification and trafficking. Ubiquitination of the activated EGFR plays a role in this downregulation. c-Cbl is a ubiquitin ligase that associates with the activated EGFR and targets it for degradation. Since E5 has been shown to form a complex with the EGFR, we tested the hypothesis that E5 affects the interaction of c-Cbl with the EGFR. We found a significant decrease of c-Cbl bound to the EGFR and of ubiquitinated EGFR in the presence of E5. E5 did not affect c-Cbl steady-state level, phosphorylation or translocation to the membrane. This novel result suggests that HPV16 E5 may, at least in part, upregulate EGFR-mediated signal transduction by inhibiting the interaction of c-Cbl with the EGFR, thereby decreasing c-Cbl-mediated degradation of the EGFR.
Human papillomavirus (HPV)type 16 (HPV16)is highly associated with cervical cancers (zur Hausen, 1996). HPV16 encodes three oncoproteins, E5, E6 and E7. E6 and E7 together are the major transforming proteins (Sedman et al.., 1991; zur Hausen, 1999; Munger et al., 2001). E6 is known to bind the tumor suppressor gene product p53 and target it for degradation (Scheffner et al., 1990; Mantovani and Banks, 1999; Thomas et al., 1999; zur Hausen, 1999). E7 degrades another tumor suppressor, pRB protein, and frees the pRB-bound E2F to activate genes necessary for S-phase entry and DNA replication (Munger et al., 1992, 2001; Gonzalez et al., 2001). The HPV16 E5 protein is weakly oncogenic: inducing anchorage-independent growth, cooperating with E7 to transform cells, enhancing the ability of E7 to induce proliferation and of E6 and E7 to immortalize cells, and contributing to the reprogramming of suprabasal cells to undergo unscheduled DNA synthesis (Leechanachai et al., 1992; Bouvard et al., 1994; Valle and Banks, 1995; Stoppler et al., 1996; Genther et al., 2003). HPV16 E5 is a small hydrophobic protein with 83 amino acids, which has been shown to localize in the cell membrane, Golgi apparatus and endosomes (Conrad et al., 1993). E5 enhances the activation of the epidermal growth factor receptor (EGFR) and its downstream signal transduction pathways (Pim et al., 1992; Straight et al., 1993; Gu and Matlashewski, 1995; Crusius et al., 1998; Zhang et al., 2002). Three mechanisms of action of HPV16 E5 have been reported: (1)E5 forms a complex with the EGFR and affects its activity (Hwang et al., 1995); (2) E5 binds the 16 kDa subunit C of the V-H+-ATPase inhibiting the acidification of late endosome, which, in turn, inhibits the degradation of endocytic EGFR and increases the recycling of the endocytic EGFR (Straight et al., 1995); and (3) E5 inhibits trafficking from early to late endosomes and delays the degradation of the activated EGFR (Thomsen et al., 2000).
The erbB family of tyrosine kinases, which includes the EGFR (erbB1), erbB2, erbB3, and erbB4, is a family of growth factor receptors, which is frequently activated in epithelial malignancies including HPV-positive cervix cancer (Kersemaekers et al., 1999; Holbro et al., 2003). EGFR is a 170 kDa transmembrane receptor tyrosine kinase (Grant et al., 2002). After ligand binding, the EGFR dimerizes and undergoes autophosphorylation on specific tyrosine residues in its cytoplasmic tail. This provides docking sites for binding of a series of tyrosine kinases and activates their downstream signaling pathways (Boni-Schnetzler and Pilch, 1987; Sorkin et al., 1991). These different signal transduction pathways are required for mitogenesis, transformation and differentiation (Hubbard, 1999). Ligand binding also triggers the internalization and finally degradation of the activated EGFR, which is the intrinsic mechanism by which cells attenuate the mitogenic signals (Di Fiore and Gill, 1999; Burke et al., 2001). Both proteasomal and lysosomal pathways are involved in this event (Levkowitz et al., 1998; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003). The lysosomal pathway involves a series of proteases in the endocytic compartment with a low pH, whereas the proteasomal pathway is related to the ubiquitination of the EGFR (Levkowitz et al., 1998; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003).
c-Cbl is a 120kDa adaptor protein, which plays an important role in the degradation of the EGFR (Levkowitz et al., 1998; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003). After EGFR activation, c-Cbl is phosphorylated by a series of tyrosine kinases and translocated to the cell membrane, where it binds the activated form of EGFR (Fukazawa et al., 1996; Levkowitz et al., 1996; de Melker et al., 2001). c-Cbl is an E3 ubiquitin ligase, which has a tyrosine kinase binding (TKB) domain, which binds the phosphotyrosine of the activated receptor as well as nonreceptor tyrosine kinases, a RING finger domain, which interacts with the ubiquitin-conjugating enzymes, E2s, and a highly proline-rich region, which binds SH3 domain-containing proteins (Fukazawa et al., 1996; Joazeiro et al., 1999; Thien and Langdon, 2001). The TKB binding domain and RING finger domain of c-Cbl allow it to mediate the ubiquitination and finally degradation of the activated EGFR (Yokouchi et al., 1999). c-Cbl-mediated ubiquitination has been shown to play an important role during EGFR degradation (Levkowitz et al., 1998; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003).
Since HPV16 E5 is a membrane protein (Conrad et al., 1993) that has been shown to form a complex with the EGFR (Hwang et al., 1995), the current study was undertaken to test the hypothesis that HPV16 E5 interferes with the interaction between c-Cbl and the EGFR. To verify that both the endocytic pathway and the proteasomal pathways regulated the EGFR in human foreskin keratinocytes (HFKs) and to compare the level of activated EGFR to that in HPV16 E5-expressing cells, HFKs were infected with parental retrovirus LXSN or recombinant retrovirus encoding HPV16 E5 [L(16E5)SN], starved for EGF and subsequently stimulated with EGF. Parallel plates of LXSN-infected cells were pretreated with MG132, a specific proteasomal inhibitor, or NH4Cl, an inhibitor of the endocytic pathway. Whole-cell lysates were analysed by Western blot using antibodies to phospho-EGFR, EGFR and GAPDH. EGFR was phosphorylated quickly after EGF binding and the level of phosphorylated EGFR rapidly decreased in LXSN-infected cells. Downregulation of activated EGFR (phosphorylated EGFR)was inhibited to a similar extent by E5, MG132 and NH4Cl (Figure 1). This result is consistent with earlier reports that downregulation of the activated EGFR involves both the lysosomal and proteasomal pathways (Levkowitz et al., 1998; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003). Also consistent with previous results, flow cytometry showed that E5-expressing keratinocytes had a greater amount of EGFR on their surface than control cells (data not shown and Straight et al., 1993).
It has been reported previously that E5 inhibits endosome acidification by binding the 16 kDa subunit C of the V-H+-ATPase (Straight et al., 1995). However, it is also possible that E5 stabilizes the activated EGFR by inhibiting its ubiquitination by c-Cbl. Binding of c-Cbl to EGFR increases when EGFR is activated at the cell membrane (Bowtell and Langdon, 1995). Since HPV16 E5 is a membrane protein and binds EGFR (Hwang et al., 1995), we hypothesized that E5 might disrupt the c-Cbl–EGFR interaction, thereby inhibiting c-Cbl-mediated EGFR degradation. To test this, whole-cell lysates were prepared from HFKs starved for and stimulated with EGF as described above. The EGFR was immunoprecipitated with antibody to EGFR. Immune complexes were separated and analysed by Western blot using antibodies to phosphotyrosine to detect activated EGFR, ubiquitin to detect ubiquitinated EGFR, EGFR and c-Cbl. Consistent with Figure 1, more phosphorylated EGFR was precipitated from E5-expressing cells. However, much less c-Cbl was associated with the EGFR (Figure 2). Since c-Cbl ubiquitinates the EGFR, decreased binding to the EGFR should result in decreased ubiquitination of the activated EGFR. This was indeed the case (Figure 2).
The disruption of c-Cbl–EGFR interaction could be due to decreased steady-state level of c-Cbl. To test whether E5 can affect the c-Cbl steady-state level, we have analysed whole-cell lysates, extracted using the same conditions as in Figure 1, by Western blot using antibodies to c-Cbl and GAPDH. E5 did not affect the c-Cbl steady-state level (Figure 3a).
During ligand-mediated EGFR activation, c-Cbl is phosphorylated and translocated to the cell membrane where it binds the activated form of EGFR, acts as a ubiquitin ligase and mediates EGFR degradation (Levkowitz et al., 1998; Joazeiro et al., 1999; Yokouchi et al., 1999; de Melker et al., 2001). To test whether the decrease in c-Cbl–EGFR interaction was due to an effect of E5 on c-Cbl phosphorylation and/or subcellular translocation during EGFR activation, the membrane fraction from LXSN- and L(16E5)SN-infected HFKs was extracted and analysed by Western blot using antibodies to c-Cbl, phospho-c-Cbl and β-actin. The results show that c-Cbl is phosphorylated and translocated to the membrane fraction similarly in control and E5-expressing cells after EGF stimulation (Figure 3b).
EGFR is one of the most important proto-oncogenes whose expression is increased during progression of cervical cancer, an HPV-associated disease (Kersemaekers et al., 1999). Overexpression of EGFR has been associated with poor prognosis (Kersemaekers et al., 1999). Exploring the mechanisms by which HPV regulates signaling by these receptors may suggest new ways to inhibit progression of HPV-mediated disease. HPV16 E6 and E7 can increase EGFR mRNA level (Akerman et al., 2001). HPV-E6/E7 and EGFR levels correlated significantly in cervical cancer and cervical intraepithelial neoplasia (CIN)(Mathur et al., 2001). The fate of the EGFR after EGF binding is normally determined in the endocytic compartment, where it is finally degraded in the lysosome or in the proteasome (Burke et al., 2001). Both pathways are important for EGFR degradation, although the relationship of the two pathways to each other in this regulation is unclear (Figure 1 and Levkowitz et al., 1998; Joazeiro et al., 1999; Yokouchi et al., 1999; de Melker et al., 2001; Longva et al., 2002; Alwan et al., 2003). Ubiquitination of EGFR has been shown to be a necessary step for EGFR degradation, where c-Cbl plays a key role (Longva et al., 2002). The EGFR must be ubiquitinated by c-Cbl in order to progress from early to late endosomes (Ravid et al., 2004). HPV16 E5 is a membrane protein, which associates with EGFR and enhances EGFR-mediated signal transduction (Pim et al., 1992; Gu and Matlashewski, 1995; Hwang et al., 1995; Crusius et al., 1998). Our findings suggest that E5 may affect the proteasomal pathway of EGFR degradation. E5 does not seem to affect the steady-state level of c-Cbl (Figure 3a)or the phosphorylation and translocation to cell membrane fraction (Figure 3b). However, while the interaction of c-Cbl and EGFR is increased when EGFR is activated in HFKs (Figure 2 and de Melker et al., 2001), this interaction and the ubiquitination of the activated EGFR is significantly decreased in the presence of E5 (Figure 2). It is possible that E5 blocks the interaction of c-Cbl with the EGFR, thereby stabilizing the activated form of EGFR and prolonging its downstream signal.
In sum, our results describe a new mechanism of action of HPV16 E5, disruption of the c-Cbl–EGFR interaction. This may contribute to the ability of E5 to decrease EGFR downregulation and prolong its activation. Since HPV16 E5 is generally considered to act early in the oncogenic process (DiMaio and Mattoon, 2001), reversal of its activity may be important to slowing the progression of HPV-mediated carcinogenesis.
The research was supported in part by a grant from the Lilly Center for Women’s Health (AR), INGEN (AR), the Thoracic Oncology Program, Indiana University (DP), a Clarian Values Foundation grant (DP)and NIH Grant 1P20-GM66402 (DP). The Indiana Genomics Initiative (INGEN) of Indiana University is supported in part by Lilly Endowment Inc. We thank Mae Lewis and Wei Chen for technical assistance and Denise Galloway for providing the L(16E5)SN packaging cell line.