| DAB2IP protein(DOC/DAB2 interactive protein) is a tumor suppressor protein associated with ovarian cancer,prostate cancer, breast cancer and choriocarinoma. It was first identified as a DOC-2/DAB2 interactive protein with several potential functional domains through a yeast two-hybrid system. The major feature of DAB2 IP protein is impact on Ras-mediated signal transduction and growth inhibition by its Ras GAP homology domain. Several research reported that the C2 domain of DAB2 IP protein binds with apoptosis signal-regulating kinase 1(ASK1), and is also called ASK1 Interacting Protein(AIP1), inhibits the binding of ASK1 and its inhibitor 14-3-3 and induces apoptosis. In addition, more and more research reported that DAB2 IP protein plays a tumor suppressor protein role in tumor growth, metastasis and tumor progression. There has some other research observed that the abnormal expression of DAB2 IP gene induced by epigenetic silencing was always detected in cancer. For example, in prostate cancer, the expression of DAB2 IP protein was inhibited by promoter methylation and histone modification. Moreover, our previous data indicated that DAB2 IP protein was degraded by E3 ubiquitin ligase SCFFbw7 in the manner of ubiquitin-proteasome dependent, thus repress its inhibition of tumor cell growth and metastasis. Given a number of research on DAB2 IP protein, there has a lot of research about the downstream signal pathway of DAB2 IP protein and the mechanism is relatively clear,however the upstream signal pathway regulatory mechanism of DAB2 IP protein is still not very clear.Ubiquitin-proteasome system is a irreversible process of proteins selective turnover and can be used by multiple cellular processes. Furthermore, many research observed that ubiquitin-proteasome system control tumor growth and metastasis through mediating the degradation of a variety of tumor related proteins. Ubiquitin-proteasome system is activated by the successive three enzymes : a ubiquitin activating enzyme(E1), ubiquitin conjugating enzyme(E2s) and ubiquitin ligase(E3s), and several research confirmed that E3 ubiquitin ligase can control the substrate specificity in ubiquitin-proteasome system. For example, E3 ubiquitin ligase SPOP degrade prostate cancer related protein ERG in a manner of ubiquitin-proteasome dependent and repress the progression of prostate cancer. In addition, there has another E3 ubiquitin ligase SCF?-TRCP degrade tumor metastasis suppressor gene-1(MTSS1) through ubiquitin-proteasome system. Thereby, ubiquitin-proteasome system may regulate many tumor-associated proteins, which can remind us the ubiquitin-proteasome system can be analyzed as the upstream regulatory mechanism of a variety of tumor-associated proteins.E3 ubiquitin-ligase Smurf1 is a HCET E3 ubiquitin ligase, is a member of Nedd4-like E3 ubiquitin ligase with oncogenic functional. In normal condition, it can bind with the PY motif of substrates through the WW domain of Smurf1 and degrade the substrate proteins through ubiquitin-proteasome system. In addition, there has other research observed Smurf1 can bind with Rho A protein and degraded it thorough the N-terminal C2 domain of Smurf1. These research indicated that in addition to Smurf1 binds and degrades substrate proteins through the classical way of the WW domain of Smurf1 recognize the PY motif of substrates, the other domains of Smurf1 may involve in the degradation process.In our previous research, we have already carefully analyze the structure and function of Smurf1 and DAB2 IP protein, and we observed that although the DAB2 IP protein doesn't have the classical PY motif of Smurf1 recognition, they have the same C2 membrane location domain, furthermore several research reported that PY motif of substrate proteins is not required for smurf1 recognizes substrate proteins. The more important thing is Smurf1 and DAB2 IP protein play an opposite role in tumor, thus it suggests that DAB2 IP protein may be binded and degraded by Smurf1 through non-classical way.Based on the analysis of structure and function of Smurf1 and DAB2 IP protein, we designed this experiment. Firstly, we detected the binding of DAB2 IP protein and Nedd4-like E3 ubiquitin ligase family to confirm the specific E3 ubiquitin ligase binding with DAB2 IP protein, and then through inhibition and overexpression of this specific E3 ubiquitin ligase to observe the regulation of this specific E3 ubiquitin ligase on the expression of DAB2 IP protein, thereby to verify whether Smurf1-mediated the DAB2 IP protein degradation via ubiquitination. Secondly, we detected whether the well-known Akt-mediated the phosphorylation of DAB2 IP protein affect the regulation of Smurf1 on DAB2 IP protein, and then analyzed whether Smurf1 has the Akt phosphorylation site and detected whether Akt-mediated Smurf1 phosphorylation further to control the expression level of DAB2 IP protein through regulate Smurf1 protein self-stability, thereby to confirm whether Akt-mediated these two proteins phosphorylation affect the process of Smurf1 control DAB2 IP protein expression level. Finally, we constructed the deletion plasmids of Smurf1 and DAB2 IP proteins(sh Smurf1 and sh DAB2IP) to make the stable cell lines(single knock-down Smurf1 or DAB2 IP and double knock-down Smurf1 and DAB2 IP in DU145 and MCF-7 cells), to explore the effect of Smurf1 control DAB2 IP protein expression level on tumor features through cell proliferation experiments(colony formation assay and soft agar assay) and migration experiment(in vitro scratch assay). Methods:Methods:(1) We used western blot and immunoprecipitation to explore the binding of DAB2 IP protein and Nedd4-like E3 ubiquitin ligases.(2) We used transient transfection to link the objective sh RNA fragment and lentiviral vector, coated virus and infected objective cells to construct stable gene knock-down cell lines to observe the effect of Smurf1 inhibition on DAB2 IP protein expression level.(3) We used transient transfection to make overexpress Smurf1 cell lines or stable knock-down Smurf1 cell lines, using protein half-life assay to detect the effect of Smurf1 on the half-life of intracellular DAB2 IP protein.(4) We used in-vivo ubiquitination assay to detect whether Smurf1 control DAB2 IP protein degradation through ubiquitination degradation pathway.(5) Transient transfection the active form of Akt and using western blot and immunoprecipitation to detect the effect of Akt on DAB2 IP and Smurf1 proteins phosphorylation level, and then detected the effect of Akt-mediated these two proteins phosphorylation on Smurf1-mediated DAB2 IP protein expression level.(6) We used transient transfection to link the objective sh RNA fragment and lentiviral vector, coated virus and infected objective cells to construct stable gene knock-down cell lines, and then used in vitro scratch assay to observe the difference of migration speed between different cell groups.(7) We used colony formation assay to detect the change of tumor cell colony formation between different cell groups.(8) We used soft agar assay to detect the change of tumor cell colony formation between different cell groups.Results:(1) The results of western blot and immunoprecipitation showed that DAB2 IP protein can combine with Smurf1 of Nedd4-like E3 ubiquitin ligase family members.(2) The inhibition of endogenous Smurf1 expression induced by sh RNA can increase DAB2 IP protein expression level in different cell lines(DU145,MCF-7 and T98 G cells). At the same time, the result of protein degradation experiment showed that the overexpression of Smurf1 can decrease DAB2 IP protein expression level.(3) The result of protein half-life experiment showed that the overexpression of Smurf1 can obviously shorten DAB2 IP protein half-life and the inhibition of Smurf1 can obviously extend DAB2 IP protein half-life.(4) The result of in-vivo ubiquitination experiment confirmed that Smurf1 can degrade DAB2 IP protein in a manner of ubiquitin-proteasome system dependent.(5) A series of protein degradation experiment and immunoprecipitation experiment showed that Akt-mediated DAB2 IP protein phosphorylation doesn't affect Smurf1 control the DAB2 IP protein expression level, however Akt-mediated the Smurf1 phosphorylation can promote Smurf1 mediated DAB2 IP protein ubiquitinationdegradation through increasing the Smurf1 self-stabilty.(6) The signal knock-down Smurf1 protein and DAB2 IP protein or double knovk-down Smurf1 and DAB2 IP proteins to construct different cells(DU45 and MCF-7 cells).The result of in vitro scratch assay showed that compared with control group, signal knock down Smurf1 can obviously inhibit cells migration speed, and the double knock-down can reduce the inhibition of signal knock-down Smurf1 on cells migration speed.(7) The results of colony formation assay and soft agar assay showed that compared with control group, signal knoc-down Smurf1 can obviously inhibit cells colony formation, and the double knock-down can reduce the inhibition of signal knock-down Smurf1 on cells colony formation.Conclusions: E3 ubiquitin-ligase Smurf1 specific binded with DAB2 IP protein and mediated DAB2 IP protein ubiquitination- degradation. Akt-mediated E3 ubiquitin ligase Smurf1 phosphorylation can increased the Smurf1 protein stability, and then control Smurf1-mediated the process of DAB2 IP protein ubiquitination-degradation. Akt-mediated Smurf1 phosphorylation and the increase of its stability can control tumor cells proliferation and migration through regulating DAB2 IP protein expression level. |
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