Control of a tumor suppressor PDCD4: Degradation mechanisms of the protein in hepatocellular carcinoma cells
Abstract
In this study, we demonstrate that EGF inhibits the TGF-β1-induced apoptosis of Huh7 cells. TGF-β1 up-regulates the expression of PDCD4 causing apoptosis, by stimulating the synthesis of PDCD4 mRNA via the Smad signaling pathway. TGF-β1 also inhibits the activation of S6 kinase 1 which phosphorylates the serine 67 residue of PDCD4 and leads to the phosphorylation of serine 71 and serine 76 in the β-TRCP binding sequence. This phosphorylation sequence causes the protein to be degraded in the ubiquitin-proteasome system. EGF activates S6 kinase 1 via the PI3K-Akt-mTOR signaling pathway and stimulates the degradation of PDCD4. EGF also suppresses PDCD4 mRNA levels. As the mTOR inhibitor rapamycin up-regulated PDCD4 mRNA levels, the PI3K-Akt-mTOR signaling pathway may control the transcription of the PDCD4 gene as well as the degradation of the protein. TPA also inhibited the TGF-β1-induced apoptosis of Huh7 cells, stimulating the degradation of the PDCD4-protein. Analyses using PDCD4 mutants with changes of serines 67, 71 and 76 to alanine revealed that the phosphorylation of serine 67 is not essential for the TPA-induced suppression of the protein. The mitogens could not suppress the PDCD4-mutant proteins with changes of serine 71 and/or serine 76 to alanine, however, indicating that phosphorylations at these residues are necessary for the proteasome-mediated degradation of PDCD4. The phosphor-mimic S71/D and S76/D mutants were able to be degraded in the ubiquitin-proteasome system unlike the mutants with changes of serine to alanine. The expression of S71/D mutant was suppressed with EGF but that of S76/D mutant was not indicating that at least partly the phosphorylation of both sites was mediated by different enzymes.
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EGF suppresses TGF-β1-induced apoptosis by decreasing PDCD4 expression.
“The results indicate that EGF suppresses TGF-β1-induced apoptosis by decreasing the PDCD4 expression.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
Rapamycin increased both the protein level of S67/D and S71/D mutants while it did not increase the protein level of S76/D mutant.
“The mTOR inhibitor rapamycin increased both the protein level of S67/D and S71/D mutants while the reagent did not increase the protein level of S76/D mutant.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
EGF and TPA suppress PDCD4 protein levels through different signaling pathways in Huh7 cells.
“From these results, it was concluded that EGF and TPA suppress PDCD4-protein levels through different signaling pathways in Huh7 cells.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
Phosphorylation at S67 is necessary for EGF-induced degradation of PDCD4 protein but is not necessary for TPA-induced degradation of the protein.
“From the results, it is concluded that the phosphorylation at S67 is necessary for the EGF-induced degradation of PDCD4-protein but is not necessary for the TPA-induced degradation of the protein.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
EGF suppresses TGF-β1-induced BAX activation, mitochondria events, caspase cascade activation, and PDCD4 expression.
“EGF suppresses the TGF-β1-induced BAX activation, mitochondria events, caspase cascade activation, and PDCD4 expression [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
EGF suppressed expression of the S71/D mutant, but not that of S67/D and S76/D mutants, in the presence of pan-PKC inhibitor.
“EGF suppressed the expression of S71/D mutant, but not that of S67/D and S76/D mutants, in the presence of pan-PKC inhibitor [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
The EGF-induced PDCD4 decrease is inhibited by inhibitors of PI3K and mTOR and by the proteasome inhibitor MG132.
“The EGF-induced PDCD4 decrease is inhibited by the inhibitors of PI3K and mTOR and by the proteasome inhibitor MG132 [66] (Figure 4).”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
All phospho-mimic mutants are degraded in the ubiquitin-proteasome system; Ser76 may be phosphorylated by S6K1 activated through the EGF-induced PI3K-Akt-mTOR signaling pathway, as well as S67 and S457, and S71 and S76 may be phosphorylated by a member of the PKC signaling pathway different from S6K1.
“Altogether, these results indicated that all these phospho-mimic mutants are degraded in the ubiquitin-proteasome system and that Ser76 may be phosphorylated by S6K1 which is activated through EGF-induced PI3K-Akt-mTOR signaling pathway, as well as S67 and S457, and that S71 and S76 may be phosphorylated by a member of PKC signaling pathway different from S6K1 (Figure 7).”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
RSK expression is low and involvement of RSK in PDCD4 protein degradation is not significant in Huh7 cells.
“Therefore, we concluded that RSK expression is low and the involvement of RSK in the PDCD4 protein degradation is not significant in the Huh7 cells.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
TPA suppresses expression of all S/D mutants both in the presence and absence of rapamycin, indicating that TPA does not activate the PKC-dependent PI3K-Akt-mTOR-S6K1 signaling pathway in Huh7 cells.
“TPA suppresses the expression of all the S/D mutants both in the presence and absence of rapamycin (Figure 5, unpublished data) indicating that TPA does not activate the PKC-dependent PI3K-Akt-mTOR-S6K1 signaling pathway in the Huh7 cells.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
S76 is phosphorylated by the EGF-induced signaling pathway and phosphorylation of S76 is inhibited by rapamycin, indicating that S76 is phosphorylated by S6K1, a substrate of mTOR.
“The results showed that S76 is phosphorylated by EGF-induced signaling pathway and the phosphorylation of S76 is inhibited by rapamycin indicating that S76 is phosphorylated by S6K1, a substrate of mTOR.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
PDCD4 protein may be phosphorylated through the EGF-activated PI3K-Akt-mTOR-S6K1 signaling pathway and degraded in the proteasome system.
“These results suggest that PDCD4-protein may be phosphorylated through the EGF-activated PI3K-Akt-mTOR-S6k1 signaling pathway and degraded in the proteasome system (Figure 4).”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
EGF showed little effect on expression of the S67/A mutant PDCD4, while TPA down-regulated expression of the mutant.
“EGF showed little effect on the expression of S67/A mutant PDCD4 as expected, while TPA down-regulated the expression of the mutant [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
The MAPK inhibitor PD98059 does not inhibit EGF- or TPA-induced suppression of PDCD4 protein levels in Huh7 cells.
“We have observed that the pathway is activated by EGF and TPA but the MAPK inhibitor PD98059 does not inhibit the EGF- or TPA-induced suppression of PDCD4-protein levels in Huh7 cells [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
All the mutants can be degraded in the ubiquitin-proteasome system and replacement of serine to glutamic acid mimics phosphorylation of serine at S67, S71, and S76.
“The results indicated that the all mutants can be degraded in the ubiquitin-proteasome system and therefore the replacement of serine to glutamic acid mimics the phosphorylation of serine at S67, S71 and S76.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
TPA may be able to induce phosphorylation of both S71 and S76 independently from phosphorylation of S67.
“Namely, TPA may be able to induce the phosphorylation of both S71 and S76 independently from the phosphorylation of S67.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
Expression levels of PDCD4 mutants S67/D, S71/D, and S76/D were low in the absence of inhibitors but upregulated by MG132 proteasome inhibitor similarly to S67/A mutants.
“On the other hand, the expression levels of the PDCD4 mutants replaced with glutamic acid (D) S67/D, S71/D and S76/D were low in the absence of inhibitors but upregulated by the treatment with MG132 proteasome inhibitor similarly to S67/A mutants.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
Both mitogens showed no effect on expression of both S71/A and S76/A mutants.
“Both mitogens showed no effect on the expression of both S71/A and S76/A mutants.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
PDCD4 mRNA levels were down-regulated by EGF treatments.
“PDCD4 mRNA levels were also down-regulated by EGF treatments.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
EGF contributes to downregulate PDCD4 expression at both levels of transcription and protein degradation and PDCD4 transcription is controlled by mTOR or its downstream signaling pathway.
“The results indicate that EGF contributes to downregulate PDCD4 expression at both levels of transcription and protein degradation and that PDCD4 transcription is controlled by mTOR or its downstream signaling pathway.”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
PDCD4 mRNA levels were not changed by TPA treatments in the hepatoma cells.
“However, the mRNA levels were not changed by TPA treatments in the hepatoma cells [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
S67 and S76 of the three serines involved in the degradation mechanism of PDCD4 protein are phosphorylated by S6K1 activated via the EGF-induced PI3K-Akt-mTOR-S6K1 signaling pathway, and S71 and S76 may be phosphorylated independently from phosphorylation of S67 by the TPA-mediated signaling pathway in Huh7 hepatoma cells.
“Altogether, it is concluded that S67 and S76 of the three serines involved in the degradation mechanism of PDCD4-protein, are phosphorylated by S6K1 activated via EGF-induced PI3K-Akt-mTOR-S6K1 signaling pathway and S71 and S76 may be phosphorylated independently from the phosphorylation of S67 by TPA mediated signaling pathway in Huh7 hepatoma cells (Figure 7).”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
Rapamycin inhibited the EGF-induced down-regulation of PDCD4 mRNA levels in Huh7 hepatoma cells.
“We have observed rapamycin inhibited the EGF-induced down-regulation of PDCD4 mRNA levels in Huh7 hepatoma cells [66].”
From Control Mechanisms of the Tumor Suppressor PDCD4: Expression and Functions (2019) · cites this paper as [66]
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References 28 · Cited by 25
- Authors
- Matsuhashi, S., Hamajima, H., Xia, J., Zhang, H., Mizuta, T., Anzai, K., & Ozaki, I.
- Affiliations
- Saga University (JP)
- First Hospital of China Medical University (CN)
- Corresponding author
- Matsuhashi, Sachiko
- Venue
- Cellular Signalling
- Publisher
- Elsevier BV
- Published
- 2014-03
- Volume
- 26
- Issue
- 3
- Pages
- 603-610
- ISSN
- 0898-6568 (ISSN-L)
- OpenAlex
- W2062376381
- Cited by
- 25
- References
- 28
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2022: 2 · 2021: 3 · 2020: 2 · 2019: 4 · 2018: 1 · 2017: 3 · 2016: 2 · 2015: 4 · 2014: 1
- FWCI
- 1.04
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Cellular Signalling (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Japan Society for the Promotion of Science (24590982)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
- Keywords
- PDCD4, tumor suppressor, protein degradation, ubiquitin-proteasome system, TGF-β1, epidermal growth factor, S6 kinase 1, Smad signaling, PI3K/AKT/mTOR pathway, rapamycin, tissue plasminogen activator, apoptosis, Huh7 cells, hepatocellular carcinoma, phosphorylation, transcriptional regulation
- MeSH terms
- Phosphoinositide-3 Kinase Inhibitors, Epidermal Growth Factor, Carcinoma, Hepatocellular, Humans, Liver Neoplasms, Mutation, Phosphorylation, RNA, Messenger, Transcription, Genetic, Signal Transduction, Transcriptional Activation, RNA-Binding Proteins, Apoptosis, Sirolimus, Ribosomal Protein S6 Kinases, 70-kDa, Cell Line, Tumor, Apoptosis Regulatory Proteins, Proto-Oncogene Proteins c-akt, Smad Proteins, Transforming Growth Factor beta1, TOR Serine-Threonine Kinases
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