GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2008) 13, 285-294. doi:10.1111/j.1365-2443.2008.01162.x
© 2008 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zheng, L.
Right arrow Articles by Ke, Y.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Zheng, L.
Right arrow Articles by Ke, Y.

E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6

 Li Zheng1,a, Huirong Ding1,a, Zheming Lu1, Yong Li1, Yaqi Pan1, Tao Ning1 and Yang Ke1,2,3,*

1 Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing 100036, China
2 Department of Cell Biology, and 3 Cancer Research Center, Peking University Health Science Center, Beijing 100083, China


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
We previously found that HPV16 E6 causes the degradation of the tumor suppressor protein TSC2, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. In the present study, we investigated the role of E6-associated protein (E6AP) in HPV16 E6-induced TSC2 degradation. Our results demonstrated that TSC2 was targeted for degradation in the presence or absence of HPV16 E6. Over-expression of E6AP enhanced the degradation of TSC2 by HPV16 E6, while expression of a dominant negative E6AP (C833A) inhibited the E6-induced degradation. Additionally, by using shRNAs to block E6AP expression in HPV16 positive and negative cells, we found a significantly prolonged TSC2 half-life. An in vivo ubiquitination assay was done to reveal that E6AP promoted the ubiquitination of TSC2 independent of HPV16 E6. We further found that TSC2 bound E6AP in the presence as well as in the absence of HPV16 E6. The binding regions on E6AP and TSC2 have been identified as amino acid (aa) 260–316, aa 428–500 and aa 1–175, aa 1251–1807, respectively. Taken together, degradation of TSC2 is mediated by E6AP ubiquitin ligase.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Tuberous sclerosis complex (TSC) is an inheritable genetic disorder characterized by the formation of benign tumors in multiple organs and is caused by mutation in either the tsc1 or the tsc2 tumor-suppressor gene (Kwiatkowski 2003). The TSC1 and TSC2 products, harmatin and tuberin, form a physical and functional complex which plays an important negative regulatory role in mTOR-mediated growth pathway (Inoki & Guan 2006). The direct downstream target of TSC2 is Rheb, a Ras family GTPase which activates mTOR leading to the phosphorylation of S6K and 4EBP1 (Gao et al. 2002b; Manning & Cantley 2003; Saucedo et al. 2003; Stocker et al. 2003; Hay & Sonenberg 2004). TSC2 suppresses Rheb activity by reducing the GTP bound Rheb level in vivo (Garami et al. 2003; Inoki et al. 2003). Phosphorylation of TSC2 by Akt inactivates TSC2 GAP activity and results in the activation of mTOR-mediated growth signal (Roux et al. 2004; Ma et al. 2005; Shaw & Cantley 2006). We have previously reported that HPV16 E6 binds and induces the degradation of the tumor suppressor TSC2 protein leading to the phosphorylation of S6 kinase even in the absence of insulin (Lu et al. 2004).

High risk human papillomaviruses (HPVs) are causative agents for cervical cancer (Bosch et al. 2002; zur Hausen 2002). Two viral proteins, E6 and E7 are expressed in nearly all cervical cancers and both are necessary and sufficient for immortalization of primary human keratinocytes in vitro indicating E6 and E7 functions are required for tumorigenesis (Hawley-Nelson et al. 1989; Munger et al. 1989; Hudson et al. 1990). The oncogenic activity of E6 and E7 results from their ability to target multiple key cellular protein functions (Mantovani & Banks 2001). Loss of function in tumor suppressors p53 and Rb accounts for the most important aspect of E6 and E7 induced transformation (Dyson et al. 1989; Munger et al. 1989; Scheffner et al. 1990; Werness et al. 1990; Heck et al. 1992). High risk HPV E6 proteins enhance the ubiquitin-mediated degradation of p53, dependent on a 100-kDa cellular protein E6-associated protein (E6AP) (Huibregtse et al. 1993a,b). E6AP is one highly conserved member of the homologous to E6AP carboxyl terminus (HECT) domain E3 ubiquitin ligase family (Huibregtse et al. 1995). In the E6AP-mediated degradation of p53 E6 determined the substrate specificity. In addition to p53, E6AP also mediates the E6-dependent proteasomal degradation of hScrib and E6TP1 (Nakagawa & Huibregtse 2000; Gao et al. 2002a).

The recent study has shown that TSC2 was targeted for degradation by the ubiquitin–proteasome pathway both in the absence and in the presence of HPV16 E6. E6AP promotes the degradation of TSC2, whereas a dominant-negative mutant of E6AP (C833A) (Talis et al. 1998) inhibits HPV16 E6-induced degradation of TSC2. Blocking E6AP expression by RNAi in HPV 16 positive cervical cancer cells and in cells without HPV infection increases the half-life of TSC2. These findings demonstrate that E6AP plays a role for TSC2 turnover under the physical condition. In case of HPV16 infection, E6 targets TSC2 for ubiquitination through E6AP and subsequent degradation by the proteasome pathway.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Ubiquitination of TSC2 in the presence and absence of HPV16 E6 in vivo

We have previously shown that HPV16 E6 promotes TSC2 degradation via proteasome pathway (Lu et al. 2004). To evaluate whether TSC2 is ubiquitinated in vivo in the presence of HPV16 E6, 293T cells were transfected with flag-tagged TSC2 COOH-terminus, which is necessary for the interaction of HPV16 E6 and TSC2 and HA-tagged ubiquitin with or without HPV16 E6. Forty-eight hours after transfection, cells were treated with the proteasome inhibitor MG132 for 4 h prior to protein extraction. The COOH-terminal portion of TSC2 was immunoprecipitated with anti-flag antibody. The ubiquitination of flag tagged TSC2 was detected by anti-HA antibody. As shown in Fig. 1, co-expression of TSC2 and ubiquitin in the absence of HPV16 E6 resulted in readily ubiquitination of TSC2 (Fig. 1, lane 3). The level of TSC2 was significantly reduced when HPV16 E6 was co-expressed (Fig. 1, lane 5, lower panel). Treatment with MG132 led to the accumulation of the ubiquitinated TSC2 both in the absence and presence of E6 (Fig. 1, lanes 4 and 6). Thus, TSC2 was ubiquitinated in vivo both basally and when destabilized by HPV16 E6.


Figure 1
View larger version (53K):
[in this window]
[in a new window]

  		Figure 1  293T cells were transfected with indicated plasmids. Forty hours after transfection, the cells were treated with MG132 (final concentration, 50 µM) or DMSO for 4 h. TSC2 C-terminus was immunoprecipitated with anti-flag antibody. The ubiquitinated TSC2 C-terminus was detected by anti-HA antibody. The lower panel represents the level of the exogenous TSC2 C-terminus.

 
Effect of E6AP on TSC2 degradation in the presence of HPV16 E6

High-risk HPV E6 proteins target several cellular proteins for degradation including p53 (Scheffner et al. 1990; Huibregtse et al. 1991, 1993a,b), hScrib (Nakagawa et al. 2000), Mcm7 (Kuhne & Banks 1998) and Bak (Thomas & Banks 1998). In the presence of E6, these proteins were associated with the HECT domain-containing Ub ligase E6AP and underwent degradation via the Ub–proteasome pathway.

To assess the role of E6AP in HPV16 E6 induced TSC2 degradation, 293T cells were transfected with TSC2 and E6 or TSC2 and E6 plus E6AP. The levels of exogenous TSC2 and E6AP were detected using the anti-HA antibody. The expression of E6 was detected with the anti-myc antibody. As shown in Fig. 2A, expression of HPV16 E6 reduced the level of TSC2 (upper panel, compare lanes 2 and 3). The expression of a dominant negative E6AP (C833A), the catalytically inactive mutant inhibited the E6-induced degradation (lanes 5).


Figure 2
View larger version (44K):
[in this window]
[in a new window]

  		Figure 2  (A) 293T cells were transfected with TSC2 C-terminus and Myc tagged HPV16 E6 plus E6AP or dominant negative E6AP, C833A. Forty-eight hours after transfection, the level of exogenous TSC2 C-terminus and E6AP were detected using anti-HA antibody. The expression of E6 was detected with anti-myc antibody. (B) HEK293T cells were transfected with indicated constructs. The expressions of myc-tagged HPV16 E6, HA-tagged E6AP and the endogenous Tuberin was determined by Western blot. The levels of Tuberin were decreased, whereas the E6AP expression level was increased. (C) 293T cells were transfected with GFP tagged-HPV16 E6 plus HA tagged E6AP or HA tagged C833A. Forty eight hours after transfection, cycloheximide was added to the final concentration of 10 µg/mL. Cells were harvested at indicated time. The expression of endogenous TSC2 was detected with anti-tuberin antibody.

 
To further investigate whether the expression of E6AP affects the stability of tuberin, an increased amount of the E6AP construct were transfected in HEK293T cells with HPV16 E6. The expressions of the endogenous tuberin were determined by a Western blot. The level of tuberin decreased, whereas the E6AP expression level increased (Fig. 2B).

Next, we determined the half-life of the endogenous TSC2 in the 293T cells over-expressing HPV16 E6 and E6AP or the dominant negative mutant E6AP, C833A. In 293T cells expressing HPV16 E6 and E6AP, TSC2 decreased 3 h after cycloheximide treatment and more than half vanished after 6 h. Contrastingly, in 293T cells co-expressing HPV16 E6 and a dominant negative E6AP, C833A, resulted in no visible degradation of TSC2 (Fig. 2C).

To confirm these results, the endogenous E6AP was blocked by RNA interference technique in Caski cells which is a human cervical carcinoma cell line reported to contain an integrated human papillomavirus type 16 genome (HPV-16, about 600 copies per cell). In Caski cells transfected with the chemically synthesized control siRNA (small interference RNA), TSC2 started to diminish 3 h after cycloheximide treatment, which was similar to what was observed in 293T cells expressing HPV16 E6. Silencing E6AP with specific siRNA in Caski cells resulted in a constant TSC2 level 12 h after cycloheximide treatment. As a control, p53 level was monitored in Caski cells with and without normal level of endogenous E6AP (Fig. 3). The delayed p53 degradation was observed when E6AP expression was blocked by siRNA. Taken together, these results indicate that E6AP is required for E6 induced TSC2 degradation.


Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		Figure 3  Caski cells were transfected with specific siRNA to E6AP or control siRNA. Cells were then treated with cycloheximide as Fig. 2. The expression of TSC2 was detected with anti-tuberin antibody. The expression of p53 in cells with normal or blocked E6AP was monitored as a control.

 
Effect of E6AP on TSC2 ubiquitination and degradation in the absence of HPV16 E6

In addition to mediating HPV E6 induced degradation, E6AP also promotes the ubiquitination and degradation of several other proteins, such as hHR23A, Blk and Mcm7 (Kuhne & Banks 1998; Kumar et al. 1999; Oda et al. 1999). We were interested in investigating whether E6AP is involved in the regulation of TSC2 ubiquitination and degradation in the absence of E6. To achieve this goal, 293T cells were transfected with siRNA to silence E6AP expression. Forty-eight hours after transfection, cells were treated with cycloheximide for the indicated hours. TSC2 and E6AP were determined at different time points by anti-tuberin and anti-E6AP antibodies respectively. A relatively high level of endogenous E6AP was seen in cells transfected with control siRNA. The expression of endogenous E6AP was efficiently silenced by siRNA (Fig. 4A). The amount of TSC2 slightly declined 6 h after cycloheximide treatment and less than half of TSC2 reduced after 12 h in the cells with normal E6AP. By contrast, TSC2 level remained constant after silencing E6AP, suggesting that E6AP is involved in the proteasome degradation of TSC2 under the physical condition (Fig. 4A).


Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		Figure 4  (A) 293T cells were transfected with E6AP specific siRNA or control siRNA. Cells were treated with cycloheximide. The level of TSC2 was detected with anti-tuberin antibody. (B) 293T cells were transfected with indicated plasmids. The endogenous TSC2 was immunoprecipitated with anti-tuberin antibody. The ubiquitinated TSC2 was then detected with anti-ubiquitin antibody. (C) 293T cells were transfected with E6AP as indicated. After 24 h, cells were cultured in 0.5% serum overnight. Cells then were treated with insulin (400 nM) for 30 min. The expression of tuberin, E6AP, S6 and phosphorylation of S6 were determined by Western blot with indicated antibody.

 
The ability of E6AP to promote TSC2 ubiquitination was then assessed. 293T cells were transfected with E6AP. The endogenous TSC2 was immunoprecipitated by the anti-tuberin antibody and detected by the anti-ubiquitin antibody. TSC2 was slightly ubiqutinated in control cells, while over-expression of E6AP strongly enhanced the ubiquitination of TSC2 (Fig. 4B). In the presence of E6, less ubiquitinated TSC2 was observed because of enhanced degradation.

To further convince that E6AP is implicated in the degradation of TSC2, we performed an experiment to examine the effects of TSC2 degradation by E6AP on its downstream signaling Fig. 4C. The data shown that degradation of TSC2 by E6AP could enhance the phosphorylation of S6 in the presence of insulin.

Interaction of TSC2 with E6AP

We next attempted to examine whether E6AP binds to TSC2. Since only HPV16 E6 induces the degradation of TSC2, we performed co-immunoprecipitation assay using HPV-16-positive Caski and HPV-18-positive HeLa cell extracts respectively. If E6AP binds to TSC2 in the presence of HPV16 E6, one would expect to see the co-precipitation of E6AP and TSC2 in Caski cells, but not in HeLa cells. Cell extracts were immunoprecipitated with specific antibodies to TSC2 and analyzed in Western blots for co-immunoprecipitation. As shown in Fig. 5, the endogenous E6AP was co-precipitated by the anti-tuberin antibody in both Caski and HeLa cells, whereas no co-precipitation was seen with the control antibody suggesting that TSC2 might interact with E6AP independent of HPV16 E6. To further define the interaction between TSC2 and E6AP, similar experiments were completed in HPV free cells. In 293T cells and HPV negative cervical cancer cell line C33A, TSC2 and E6AP were also co-precipitated (Fig. 5). These results indicate that TSC2 and E6AP physically interact independent of HPV16 E6.


Figure 5
View larger version (42K):
[in this window]
[in a new window]

  		Figure 5  The endogenous TSC2 in different cells as labeled was immunoprecipitated with anti-tuberin antibody. The presence of E6AP in the complex was detected with anti-E6AP antibody.

 
E6AP binding domain for TSC2

The E6AP binding domain for TSC2 was investigated. In our experiment the E6AP is not a full-length clone but rather an N-terminal truncation that encodes a 95-kDa version of the protein, because the full-length clone translated poorly in vitro (Huibregtse et al. 1993a). A series of E6AP deletions were fused to GST and expressed in Escherichia coli. GST pull-down assays found that the carboxyl-terminal deletions of E6AP (43–316), E6AP (43–407) and E6AP (43–430) but not E6AP (43–260), and the amino-terminal deletions of E6AP (428–875), and E6AP (448–875), but not E6AP (500–875), were able to bind to TSC2 (Fig. 6A). These results suggest that the regions from aa 260 to aa 316 and from aa 428 to aa 500 are important for TSC2 binding. TSC2 interacts with HPV16 E6 via L2G box. There is a L2G box in E6AP. The deletion experiments demonstrated that the L2G motif was not required for TSC2 and E6AP interaction. To further confirm this observation, the L2G box was deleted from GST-E6AP (43–460). The resulting fusion protein retained the ability to be bound to TSC2.


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		Figure 6  (A) The deletions of E6AP were illustrated on the top panel. E6AP deletions were expressed as GST fusion proteins. The TSC2 was in vitro transcript-translated in a wheat germ-cell free system. The binding assay was carried out under the condition described in Experimental procedures section. The presence of TSC2 on GST-E6AP beads was detected with anti-tuberin antibody. (B) The deletions of TSC2 were illustrated on the top panel. Flag-tagged deletions were synthesized in vitro. The binding of GST-E6AP fusion protein with flag-tagged TSC2 deletions was carried out. The presence of TSC2 deletions on GST-E6AP beads was detected with anti-flag antibody.

 
TSC2 binding domain for E6AP

A panel of TSC2 deletion mutants was used to investigate the region of TSC2 for E6AP binding. GST-E6AP was found to co-precipitate with FLAG-TSC2-N1 (1–175), FLAG-TSC2-N4 (966–1323) and FLAG-TSC2-C (1315–1807) but not with FLAG-TSC2-N2 (176–576) and FLAG-TSC2-N3 (572–965) deletions (Fig. 6B). No association of control GST protein with any FLAG-TSC2 deletions was observed. Because there is an overlap between FLAG-TSC2-N4 (966–1323) and FLAG-TSC2-C (1315–1807), we constructed a series of mutants for further study. We found that FLAG-TSC2 (1251–1400) and FLAG-TSC2 (1401–1807) could interact with GST-E6AP, but FLAG-TSC2 (966–1250) could not. This data suggests that the regions from aa 1 to aa 175 and aa 1251 to aa 1807 of TSC2 are required for E6AP binding.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have previously demonstrated that HPV16 E6 interacts with the tumor suppressor TSC2 and promotes its degradation, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. It is well documented that the HECT domain-containing ubiquitin ligase E6AP is involved in high risk HPV E6 induced cellular protein degradation. Additionally, E6AP promotes the ubiquitination of cellular proteins in the absence of E6, indicating that E6AP might function as an E3 enzyme independent of E6 (Kuhne & Banks 1998; Thomas & Banks 1998; Harris et al. 1999; Oda et al. 1999; Scheffner & Whitaker 2003). We therefore investigated the role of E6AP in TSC2 degradation both in the presence and absence of HPV16 E6. In the present study, we have shown that TSC2 was ubiquitinated and degraded through E6AP-dependent proteolysis pathway, while HPV16 E6 further accelerates TSC2 turnover. Several lines of evidences from our experiments support the above notion. First, the ubiquitinated TSC2 was readily observed both in the presence or absence of HPV16 E6 protein. In the presence of E6, less ubiquitinated TSC2 was observed because of enhanced degradation. MG132 treatment inhibited the degradation of TSC2, even in the absence of HPV16 E6, indicating that similar to other E6 targets such as p53, TSC2 levels are normally regulated by the Ub–proteasome pathway. Second, down-regulation of E6AP function by siRNA both in HPV16 positive and negative cells increases TSC2 half-life, while the half-life of TSC2 is longer in the absence of HPV16 E6 with normal E6AP. Co-expression of E6 and C833A, a dominant negative mutant E6AP, eliminates the ability of E6 to induce TSC2 degradation indicating a principal role of E6AP in HPV16 E6 induced TSC2 degradation. Finally, TSC2 and E6AP were associated in the cells without HPV16 E6 suggesting that E6AP may play a role in TSC2 turnover even in the absence of HPV16 E6.

E6AP is the prototype of HECT domain E3 ubiquitin ligase family. The members of this family are large proteins with a conserved 350-amino acid carboxyl-terminal catalytic domain, in which ubiquitin-thioester formation occurs at the active cysteine residue (Huibregtse et al. 1995). The amino-terminal domains in HECT E3 ligases are generally divergent and may provide the regions for substrate recognition. The TSC2 binding region of E6AP was mapped to two fragments located from aa 260 to aa 316 and from aa 428 to aa 500. A L2G box (TLQELLG) was previously found to interact with HPV E6 and Mcm7 (Kuhne & Banks 1998). Our data indicated that the E6 binding region containing the L2G motif is not required for the interaction between TSC2 and E6AP.

The complexity of TSC1/TSC2 plays an important role in multiple cellular functions including cell growth control and cell cycle progression (Soucek et al. 1997, 1998a,b; Miloloza et al. 2000; Rosner et al. 2003, 2004; Inoki & Guan, 2006). TSC2 is a GTPase activating protein toward Rheb and negatively controls the mTOR signaling whereas TSC1 to bind and stabilize TSC2. The recent study demonstrated that a HECT E3 ligase, HERC1, interacts with TSC2 at its NH2-domain. TSC1 inhibits the interaction between TSC2 and HERC1 under normal condition but failed to disrupt the interaction between HERC1 and TSC2 mutants found in TSC patients suggesting the involvement of HERC1 in the pathological pathway of TSC (Chong-Kopera et al. 2006). In this report, we defined three amino acid regions of TSC2 that are involved in the association with E6AP. One of the binding regions is located in the NH2-terminus of TSC2 and two are located in the COOH-terminus part of TSC2. Since the NH2-teminus of TSC2 interacts with TSC1, which may stabilize TSC2 under normal conditions, it would be important to investigate whether E6AP interferes with the TSC1 function under normal conditions and whether E6AP plays a role in the pathological pathway of TSC similar to HERC1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmids, shRNAs, antibodies and reagents

pMT123 encoding 8x HA-ubiquitin was a gift from Dr Dirk Bohmann (European Molecular Biology Laboratory, Germany University of Rochester). pGEX-2T-E6AP, pCMV4-HA E6AP and pCMV4-HA C833A were kindly provided by Dr Peter M Howley (Harvard Medical School, Boston, MA). The HPV16 full-length plasmid, the full-length TSC2 expression vector with Xpress tag, pEGFP-N1, pEGFP-16E6, pCMV-Myc-16E6, pCMV-HA-TSC2-C and pCDNA3-2 x flag-TSC2-C were described previously (Lu et al. 2004). pCDNA3-2 x flag-TSC2 mutants were amplified from TSC2 cDNA clone using the following primer pairs:

5'CGA ATT CCG GCC AAA CCA ACA AGC and 5'CCG CTC GAG TTC CGA GGA CAA GCC AAC for TSC2-N1, 5' CGA ATT CCG TTC CTT CTG GTG and 5'CCG CTC GAG GTA CAG CTT GGT CTG for TSC2-N2, 5' CGA ATT CCG CAG ACC AAG CTG TAC and 5' CCG CTC GAG TTC TTT CAC GGG TGG AG for TSC2-N3, 5' CGA ATT CCG TTC AAG GAG AGC TCT and 5'CCG CTC GAG TAG CGC TGC CTC AAC for TSC2-N4. 5' CGA ATT CCG TTC AAG GAG AGC TCT and 5'CCG CTC GAG GTA CAG GGC TGT GTC CCG for TSC2 (966–1250), 5' CGA ATT CCG AAG TCA CTG TCG GTG C and 5'CCG CTC GAG GTC CCC GAG GAT GTC CTG for TSC2 (1251–1400), 5' CGA ATT CCG CCT GGG GAC AAG G and 5'CCG CTC GAG CAC AAA CTC GGT GAA for TSC2 (1401–1807).

E6AP mutants were subcloned into pGEX-4T-2 at Sal I/Not I sites. The following primers were used: 5'AC GCG TCG ACT GAA GCC TGC ACG AAT GAG and 5'AAG GAA AAA AGC GGC CGC TTA ATT GAG AAA GGC AG for E6AP-N (43–260), 5'AC GCG TCG ACT GAA GCC TGC ACG AAT GAG and 5' AAG GAA AAA AGC GGC CGC TTA ATC ATT CAC TAG ATT TCG for E6AP-N (43–361), 5'ACGCG TCG ACT GAA GCC TGC ACG AAT GAG and 5'AAG GAA AAA AGC GGC CGC TTA CTG AAG TGT CAG CTC GCT for E6AP-N (43–407), 5'AC GCG TCG ACT AAT GAA CCA CTG AAT GAG G and 5' AAG GAA AAA AGC GGC CGC TTA TAT GCC AAT CAG AGT for E6AP-C448, 5'AC GCG TCG ACT GAA CGA AGA ATC ACT G and 5' AAG GAA AAA AGC GGC CGC TTA TAT GCC AAT CAG AGT for E6AP-C500. 5'ACGCG TCG ACT GAA GCC TGC ACG AAT GAG and 5'AAG GAA AAA AGC GGC CGC TTA ATC TTT ATC CAT TTC for E6AP-N (43–460), Up1 5'AC GCG TCG ACT GAA GCC TGC ACG AAT GAG, Down1 5'TTC TTG TTT CTT CTT TCT TC C AGC TCG CT, Up2 5'CAG CGA GCT GGA AGA AAG AAG AAA C, Down2 5'AAG GAA AAA AGC GGC CGC TTA ATC TTT ATC CAT TTC for E6AP-N (43-460{Delta}L2G). E6AP-N (43–430) and E6AP-C (428-end) were first amplificated from E6AP full length ORF and subcloned in the plasmid pCMV-HA at KpnI/NotIsites and were then reconstructed into pGEX-4T-2 at SalI/NotIsites. The primers are: 5'GG GGT ACC GAA GCC TGC ACG AAT GAG and 5'AAG GAA AAA AGC GGC CGC TTA ACC AAG TTC AGT TTC CAG GGG for E6AP-N (43–430), 5'GG GGT ACC CTT GGT GTT AAA ACC CTG GAT TG and 5'AAG GAA AAA AGC GGC CGC TTA CAG CAT GCC AAA TCC for E6AP-C (428-end). All constructs generated by PCR were confirmed by DNA sequencing. Plasmids used for transfection were purified by the Qiagen (Valencia, CA) plasmid mega kits. The E6AP shRNA targeted sequence cited from Kelley et al. (2005) and control were synthesized by Shanghai GeneChem Co. (Shanghai, China), which were as follows: E6AP A, 5'CAACUCCUGCUCUGAGAUAtt and non-silencing: UUCUCCGAACGUGUCACGUtt.

Anti-tuberin (C20), anti-E6AP, anti-HA-probe (F-7), anti-P53 (DO-1), anti-actin and anti-GFP were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG antibody was from Sigma (St Louis, MO). Anti-phospho-S6 Ribosomal protein (Ser235/236, anti-S6 Ribosomal protein and anti-ubiquitin (P4D1) antibodies were from Cell Signaling Inc. (Beverly, MA) Anti-Myc antibody was from Invitrogen (Carlsbad, CA).

Proteasome inhibitor MG132 (carbobenzoxy-L-leuxyl-L-leucinal) was purchased from Calbiochem (La Jolla, CA). Cycloheximide was from Sigma.

Cell culture, transfection and immunoprecipitation

HPV-16-positive Caski and Siha, HPV-18-positive HeLa, HPV-negative C33A human cervical carcinoma cell lines, and 293T were all maintained in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum at 37 °C and 5.4% CO2. Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The total amount of plasmid DNAs were added to each plate and kept constant by including the appropriate amount of empty vector. Cells were lysed in RIPA buffer and immunoprecipitated with the indicated antibodies and protein G-Sepharose beads. Immunocomplexes were subjected to SDS-PAGE and Western blot was performed with anti-tuberin or anti-E6AP antibodies.

In vivo ubiquitination assays

293T cells (1 x 106 per 100-mm dish) were transfected with 2 µg of pCDNA3.0-2 x FLAG-TSC2-C, 2 µg pMT123 with or without 4 µg of pCMV-myc-16E6 using Lipofectamine 2000. Each dish was co-transfected with 500 ng of GFP to control for transfection efficiency. Forty hours after transfection, the cells were treated with MG132 (final concentration, 50 µM) or DMSO for 4 h. Cell lysates were then prepared in RIPA buffer. Immunoprecipitation was done as manufacturer's instructions (Amersham Pharmacia, Uppsala, Sweden) with anti-flag and immunoblotted with anti-HA. Signals were then detected using the enhanced chemiluminescence (ECL) method, as suggested by manufacturer (Amersham Biosciences Inc., Piscataway, NJ). Flag-TSC2-C was detected on parallel blots with anti-flag antibody.

In vitro binding assays

Expression of GST-fusion proteins was carried out according to manufacturer's instructions (Amersham Pharmacia). Equivalent amounts of sepharose-bound GST fusion proteins were combined with 15 µL in vitro expressed proteins. Reactions were rotated at 4 °C for 3 h in 20 mM Tris–HCl, pH 7.5, 125 mM NaCl, 75 nM EDTA, 0.08% NP-40, 1 mM PMSF, then washed 3 times with 1xPBS, pH 7.4, 1% Triton X-100, 1 mM PMSF. Samples were analyzed by SDS-PAGE. Western blots were performed by anti-tuberin or flag antibodies as indicated.

In vivo E6-induced TSC2 degradation in the presence of E6AP

To assess the effect of E6AP or its mutant on TSC2 degradation in vivo, 0.5 µg of pCMV-TSC2-C was co-transfected into 3 x 105 293T cells per 60-mm plate together with 1 µg of pCMV4 constructs encoding wild-type E6AP or 1 µg of its dominant negative mutant (C833A) with or without 1 µg of pCMV-myc-16E6. Each plate was also co-transfected with 0.1 µg of GFP for transfection efficiency control. The total amount of DNA was balanced with empty vector to keep an equal amount. After 48 h, cell lysates were prepared in sample buffer, and TSC2-C was detected by Western blot using an anti-HA antibody. HPV16 E6 or E6AP proteins was detected using anti-myc or anti-HA antibodies. GFP was detected using anti-GFP antibody.

E6AP induced TSC2 ubiquitin in vivo

A total of 3 x 105 293T cells were transfected with 3 µg E6AP in the presence or absence of 3 µg HPV 16 E6. Forty eight hours after transfection, cells were lyzed in RIPA. Immunoprecipitation was done with anti-tuberin and immunoblotted with anti-ubiquitin. Signals were then detected using the ECL.

Half-life of tuberin

Caski and 293T cells were transfected with shRNA or control at 20 nm, 48 h later, 10 µg/mL cycloheximide was added for the indicated time. Then cells were harvested for Western blot.


   Acknowledgements
 
This study was supported by Natural Science Foundation of China (30430710, 30400542), High Technology Research "863" Key Project 2006AA02Z467 and 2006AA02A403 to Yang Ke.


   Footnotes
 
Communicated by: Masayuki Yamamoto (The University of Tokyo)

aThese authors equally contributed to the paper. Back

* Correspondence: E-mail: keyang{at}bjmu.edu


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bosch, F.X., Lorincz, A., Munoz, N., Meijer, C.J. & Shah, K.V. (2002) The causal relation between HPV and cervical cancer. J. Clin. Pathol. 55, 244–265.[Abstract/Free Full Text]

Chong-Kopera, H., Inoki, K., Li, Y., Zhu, T., Garcia-Gonzalo, F.R., Rosa, J.L. & Guan, K.L. (2006) TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. J. Biol. Chem. 281, 8313–8316.[Abstract/Free Full Text]

Dyson, N., Howley, P.M., Munger, K. & Harlow, E. (1989) The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934–937.[Abstract/Free Full Text]

Gao, Q., Kumar, A., Singh, L., Huibregtse, J.M., Beaudenon, S., Srinivasan, S., Wazer, D.E., Band, H. & Band, V. (2002a) Human Papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase1. Cancer Res. 62, 3315–3321.[Abstract/Free Full Text]

Gao, X., Zhang, Y., Arrazola, P., Hino, O., Kobayashi, T., Yeung, R.S., Ru, B. & Pan, D. (2002b) TSC tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4, 699–704.[CrossRef][Medline]

Garami, A., Zwartkruis, F.J.T., Nobukuni, T., Joaquin, M., Roccio, M., Stocker, H., Kozma, S., Hafen, E., Bos, J.L. & Thomas, G. (2003) Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11, 1457–1466.[CrossRef][Medline]

Harris, K.F., Shoji, I., Cooper, E.M., Kumar, S., Oda, H., & Howley, P.M. (1999) Ubiquitin-mediated degradation of active Src tyrosine kinase. Proc. Natl. Acad. Sci. USA 96, 13738–13743.[Abstract/Free Full Text]

Hawley-Nelson, P., Vousden, K.H., Hubbert, N.L., Lowy, D.R. & Schiller, J.T. (1989) HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 3905–3910.[Medline]

Hay, N. & Sonenberg, N. (2004) Upstream and downstream of mTOR. Genes Dev. 18, 1926–1945.[Abstract/Free Full Text]

Heck, D.V., Yee, C.L., Howley, P.M. & Munger, K. (1992) Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses. Proc. Natl. Acad. Sci. USA 89, 4442–4446.[Abstract/Free Full Text]

Hudson, J.B., Bedell, M.A., McCance, D.J. & Laiminis, L.A. (1990) Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J. Virol. 64, 519–526.[Abstract/Free Full Text]

Huibregtse, J.M., Scheffner, M., Beaudenon, S. & Howley, P. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92, 2563–2567.[Abstract/Free Full Text]

Huibregtse, J.M., Scheffner, M. & Howley, P.M. (1991) A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 10, 4129–4135.[Medline]

Huibregtse, J.M., Scheffner, M. & Howley, P.M. (1993a) Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13, 775–784.[Abstract/Free Full Text]

Huibregtse, J.M., Scheffner, M. & Howley, P.M. (1993b) Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53 ubiquitination of associated proteins. Mol. Cell. Biol. 13, 4918–4927.[Abstract/Free Full Text]

Inoki, K. & Guan, K. (2006) Complexity of the TOR signaling network. Trends Cell Biol. 16, 206–212.[CrossRef][Medline]

Inoki, K., Li, Y., Xu, T. & Guan, K.L. (2003) Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834.[Abstract/Free Full Text]

Kelley, M.L., Keiger, K.E., Lee, C.J. & Huibregtse, J.M. (2005) The global transcriptional effects of the human papillomavirus E6 protein in cervical carcinoma cell lines are mediated by the E6AP ubiquitin ligase. J. Virol. 79, 3737–3747.[Abstract/Free Full Text]

Kuhne, C. & Banks, L. (1998) E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box. J. Biol. Chem. 273, 34302–34309.[Abstract/Free Full Text]

Kumar, S., Talis, A.L. & Howley, P.M. (1999) Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination. J. Biol. Chem. 274, 18785–18792.[Abstract/Free Full Text]

Kwiatkowski, D.J. (2003) Tuberous sclerosis: from tubers to mTOR. Ann. Hum. Genet. 67, 87–96.[CrossRef][Medline]

Lu, Z., Hu, X., Li, Y., Zheng, L., Zhou, Y., Jiang, H., Ning, T., Basang, Z., Zhang, C. & Ke, Y. (2004) Human papillomavirus 16 E6 oncoprotein interferences with insulin signaling pathway by binding to tuberin. J. Biol. Chem. 279, 35664–35670.[Abstract/Free Full Text]

Ma, L., Chen, Z., Erdjument-Bromage, H., Tempst, P. & Pandolfi, P.P. (2005) Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 121, 179–193.[CrossRef][Medline]

Manning, B.D. & Cantley, L.C. (2003) Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576.[CrossRef][Medline]

Mantovani, F. & Banks, L. (2001) The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 20, 7874–7887.[CrossRef][Medline]

Miloloza, A., Rosner, M., Nellist, M., Halley, D., Bernaschek, G. & Hengstschläger, M. (2000) The TSC1 gene product, hamartin, negatively regulates cell proliferation. Hum. Mol. Genet. 9, 1721–1727.[Abstract/Free Full Text]

Munger, K., Werness, B.A., Dyson, N., Phelps, W.C., Harlow, E. & Howley, P.M. (1989) Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8, 4099–4105.[Medline]

Nakagawa, S. & Huibregtse, J.M. (2000) Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol. Cell. Biol. 20, 8244–8253.[Abstract/Free Full Text]

Oda, H., Kumar, S. & Howley, P. (1999) Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. Proc. Natl. Acad. Sci. USA 96, 9557–9562.[Abstract/Free Full Text]

Rosner, M., Freilinger, A. & Hengstschläger, M. (2004) Proteins interacting with the tuberous sclerosis gene products. Amino Acids 27, 119–128.[CrossRef][Medline]

Rosner, M., Hofer, K., Kubista, M. & Hengstschläger, M. (2003) Cell size regulation by the human TSC tumor suppressor proteins depends on PI3K and FKBP38. Oncogene 22, 4786–4798.[CrossRef][Medline]

Roux, P.P., Balliff, B.A., Anjum, R., Gygi, S.P. & Blenis, J. (2004) Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc. Natl. Acad. Sci. USA 101, 13489–13494.[Abstract/Free Full Text]

Saucedo, L.J., Gao, X., Chiarelli, D.A., Li, L., Pan, D. & Edgar, B.A. (2003) Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5, 566–571.[CrossRef][Medline]

Scheffner, M., Werness, B.A., Huibregtse, J.M., Levine, A.J. & Howley, P.M. (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129–1136.[CrossRef][Medline]

Scheffner, M. & Whitaker, N.J. (2003) Human papillomavirusinduced carcinogenesis and the ubiquitin-proteasome system. Semin. Cancer Biol. 13, 59–67.[CrossRef][Medline]

Shaw, R.J. & Cantley, L.C. (2006) Ras, PI3K and mTOR signalling controls tumour cell growth. Nature 441, 424–430.[CrossRef][Medline]

Soucek, T., Hölzl, G., Bernaschek, G. & Hengstschläger, M. (1998a) A role of the tuberous sclerosis gene-2 product during neuronal differentiation. Oncogene 16, 2197–2204.[CrossRef][Medline]

Soucek, T., Pusch, O., Wienecke, R., DeClue, J.E. & Hengstschläger, M. (1997) Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J. Biol. Chem. 272, 29301–29308.[Abstract/Free Full Text]

Soucek, T., Yeung, R.S. & Hengstschläger, M. (1998b) Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc. Natl. Acad. Sci. USA 95, 15653–15658.[Abstract/Free Full Text]

Stocker, H., Radimerski, T., Schindelholz, B., Wittwer, F., Belawat, P., Daram, P., Breuer, S., Thomas, G. & Hafen, E. (2003) Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5, 559–565.[CrossRef][Medline]

Talis, A.L., Huibregtse, J.M. & Howley, P.M. (1998) The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells. J. Biol. Chem. 273, 6439–6445.[Abstract/Free Full Text]

Thomas, M. & Banks, L. (1998) Inhibition of Bak-induced apoptosis by HPV-18 E6. Oncogene 17, 2943–2954.[CrossRef][Medline]

Werness, B.A., Levine, A.J. & Howley, P.M. (1990) Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76–79.[Abstract/Free Full Text]

zur Hausen, H. (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2, 342–350.[CrossRef][Medline]

Received: 23 April 2007
Accepted: 5 December 2007





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zheng, L.
Right arrow Articles by Ke, Y.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Zheng, L.
Right arrow Articles by Ke, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS