SGEF增强EGFR蛋白稳定性的分子机制及生物学意义
发布时间：2019-07-06 17:48
【摘要】：EGFR信号通路的异常激活与前列腺癌的恶性进展、高Gleason分级及雄激素非依赖进程密切相关。最新的研究发现，在前列腺肿瘤标本中，仅有8%的EGFR发生突变，但却有高达31%的标本中EGFR发生高表达，提示EGFR的高表达是前列腺癌中EGFR信号通路异常激活的主要原因。EGFR在肿瘤组织中高表达主要有两个原因，一是基因扩增，另一个是蛋白降解受阻。该项研究并没有发现组织水平EGFR高表达与EGFR基因扩增具有显著的相关性，提示EGFR蛋白分子的降解受阻可能是前列腺癌中EGFR异常表达的主要原因。但目前对于前列腺癌中EGFR蛋白稳定性调控的分子机制还不明确，因此发现新的EGFR蛋白稳定性的调节分子对于阐明前列腺癌恶性进展的分子机制具有重要意义。SGEF是我们前期发现的前列腺癌中的一种潜在的癌基因，临床研究表明SGEF随着前列腺癌的恶性进展表达水平逐渐升高，且我们发现SGEF能够促进前列腺癌细胞的增殖和存活能力，但我们对于SGEF在前列腺癌中发挥癌基因作用的具体分子机制还不清楚。我们的前期研究发现，敲低SGEF能够抑制AKT分子的磷酸化，而AKT是EGFR下游的重要分子，因此我们推测SGEF有可能通过对EGFR蛋白稳定性的调节进而调控EGFR/AKT信号通路。 我们首先检测了SGEF是否影响前列腺癌细胞中EGFR蛋白表达水平，我们发现敲低SGEF降低了前列腺癌细胞C4-2B和DU145中EGFR的蛋白表达水平，提示我们SGEF确实能够调控前列腺癌细胞中EGFR蛋白表达水平。为了进一步确定SGEF调控EGFR蛋白表达水平的机制，我们检测了过表达SGEF对EGFR蛋白降解的影响，发现在293T细胞和COS7细胞中过表达SGEF能够明显EGFR蛋白的降解。随后我们在多种前列腺癌细胞中检测了敲低SGEF对EGFR蛋白降解的影响，我们发现在多种前列腺癌细胞中敲低SGEF均能明显促进EGFR蛋白的降解。这些结果说明SGEF能够通过增强EGFR蛋白稳定性来增强前列腺癌细胞中EGFR的蛋白表达水平。由于SGEF是RhoG特异性的鸟苷酸交换酶，，我们随后检测了SGEF是否以鸟苷酸交换酶活性依赖的方式增强EGFR蛋白的稳定性。我们缺失鸟苷酸交换酶活性的SGEF依然能够抑制EGFR蛋白的降解，同时发现持续激活型的RhoG不能够回转敲低SGEF对EGFR蛋白降解的促进作用，这些结果说明SGEF是以鸟苷酸交换酶活性非依赖的方式增强EGFR蛋白的稳定性。 EGFR在细胞中的降解受多重调控，EGFR与配体结合后，发生二聚化和自磷酸化，磷酸化的EGFR招募E3泛素酶Cbl促使EGFR发生泛素化。泛素化的EGFR在Eps15和epsin-1的介导下发生内吞进入细胞浆中。内吞的EGFR先进入早期内体，然后在ESCRT复合物介导下进入晚期内体，最后在溶酶体中发生降解。我们随后检测了SGEF是通过哪一环节参与EGFR蛋白的降解调节。我们发现过表达SGEF并不影响EGFR的泛素化、内吞和进入早期内体，而是抑制EGFR从早期内体进入晚期内体。随后我们在前列腺癌细胞中发现敲低SGEF不影响EGFR进入早期内体，而是促进了EGFR从早期内体进入晚期内体。这些结果说明了SGEF是通过抑制EGFR从早期内体进入晚期内体进而抑制EGFR蛋白降解的。 EGFR的高表达通常与EGFR信号通路的异常激活密切相关，我们随后检测了SGEF是否能够调控EGFR信号通路。我们发现敲低SGEF明显抑制了EGF诱导的EGFR和下游蛋白AKT的磷酸化。由于EGFR/AKT信号通路在前列腺癌细胞迁移中发挥着重要作用，我们利用划痕实验检测了SGEF对前列腺癌细胞迁移的影响，结果发现敲低SGEF明显抑制了EGF诱导的细胞迁移。 EGFR/ERK1/2信号通路在细胞多种生命活动中均发挥着重要作用。既然我们发现了SGEF能够增强EGFR蛋白的稳定性，我们随后检测了SGEF对EGFR/ERK1/2信号通路的影响。我们发现过表达SGEF能够显著增强EGF诱导的ERK1/2磷酸化，而敲低SGEF能够明显抑制EGF诱导的ERK1/2磷酸化，说明SGEF能够增强EGFR/ERK1/2信号通路。随后我们检测了SGEF是否以RhoG依赖的方式增强EGFR/ERK1/2信号通路，我们发现敲低RhoG的表达并不能抑制SGEF对EGFR/ERK1/2信号通路的增强作用，说明SGEF能够以RhoG非依赖的方式增强EGFR/ERK1/2信号通路。 Grb2在EGF诱导的ERK1/2激活的过程中发挥着重要的作用。由于Grb2的两端含有SH3结构域，而SGEF的N端含有Pro结构域，而SH3结构域与Pro结构域之间存在着经典的相互作用，因此我们推测SGEF有可能通过与Grb2相互作用进而增强EGFR/ERK1/2信号通路。我们通过GST-pull down实验和免疫共沉淀实验验证了两者之间的相互作用，随后确认了Grb2的SH3结构域与SGEF的Pro结构域介导了两者之间的相互作用。但当我们检测缺失与Grb2相互作用的SGEF突变体对EGFR/ERK1/2信号通路的影响时，我们出乎意料的发现与野生型SGEF相比，SGEF突变体不但没有减弱对EGFR/ERK1/2信号通路的增强作用，反而进一步增强了EGF诱导的ERK1/2磷酸化，这就提示我们Grb2可能通过与SGEF相互作用拮抗SGEF对EGFR/ERK1/2信号通路的增强作用。随后我们发现过表达Grb2确实能够抑制SGEF对EGFR/ERK1/2信号通路的增强作用，而敲低Grb2促进了SGEF对EGFR/ERK1/2信号通路的增强作用，并且发现Grb2拮抗SGEF对EGFR/ERK1/2信号通路的增强作用依赖与Grb2与SGEF之间的相互作用。 总之，本研究发现了SGEF能够通过抑制EGFR从早期内体进入晚期内体从而增强EGFR蛋白稳定性。由于EGFR的高表达是前列腺癌细胞中EGFR信号通路异常激活的主要原因，SGEF对于EGFR蛋白稳定性的调控可能是SGEF促进前列腺癌恶性进展的原因之一。同时由于多种生长因子受体的降解方式与EGFR一致，SGEF有可能通过类似的方式抑制多种生长因子受体的降解，发挥其癌基因的功能。同时我们发现SGEF能够以RhoG非依赖的方式增强EGF诱导的ERK1/2激活，且Grb2能够通过与SGEF相互作用拮抗SGEF对EGFR/ERK1/2信号通路的增强作用。由于Grb2一直被认为在EGFR/ERK1/2信号通路中发挥正调控作用，本研究中发现了在SGEF过表达的情况下Grb2能够负调控EGFR/ERK1/2信号通路，揭示出Grb2在EGFR/ERK1/2信号通路调控中功能的复杂性。
[Abstract]:The abnormal activation of the EGFR signaling pathway is closely related to the malignant progression of prostate cancer, the high Gleason classification and the androgen-independent process. The most recent study found that only 8% of the EGFR mutations in the prostate tumor specimens, but up to 31% of the samples, showed high EGFR expression, suggesting that the high expression of EGFR was the main cause of the abnormal activation of the EGFR signaling pathway in prostate cancer. The high expression of EGFR in tumor tissue is mainly two reasons, one is gene amplification, and the other is protein degradation. The study did not find a significant correlation between the high expression of EGFR and the amplification of EGFR gene, suggesting that the inhibition of EGFR protein molecule could be the main cause of the abnormal expression of EGFR in prostate cancer. However, the molecular mechanism of the stability and control of EGFR protein in prostate cancer is not clear, and therefore, it is of great significance to find the molecular mechanism of the stability of the new EGFR protein. SGEF is a potential oncogene in the early stage of prostate cancer, and the clinical study shows that the level of SGEF is gradually increasing with the malignant progression of prostate cancer, and we find that the SGEF can promote the proliferation and survival ability of prostate cancer cells. But the specific molecular mechanism of the role of SGEF in prostate cancer is unclear. Our previous studies have found that the knockdown of SGEF can inhibit the phosphorylation of AKT molecules, and AKT is an important molecule downstream of the EGFR, so we assume that the SGEF is likely to control the EGFR/ AKT signal pathway by modulating the stability of the EGFR protein. We first examined whether the SGEF has an effect on the level of EGFR protein expression in prostate cancer cells, and we have found that the knockdown of SGEF reduces the level of EGFR protein expression in prostate cancer cells C4-2B and DU145, suggesting that the SGEF is indeed capable of regulating the expression of EGFR protein in prostate cancer cells In order to further determine the mechanism of SGEF to regulate the level of EGFR protein expression, we have examined the effect of overexpression of SGEF on the degradation of EGFR protein, and it was found that overexpression of SGEF in 293T cells and COS7 cells could significantly reduce the decrease of EGFR protein. Solutions. Then we tested the effect of knock-on low SGEF on the degradation of EGFR protein in a variety of prostate cancer cells, and we found that the knockdown of the low SGEF in a variety of prostate cancer cells could significantly contribute to the reduction of the EGFR protein. These results indicate that the SGEF can enhance the protein expression of EGFR in prostate cancer cells by enhancing the stability of the EGFR protein Ping. Since the SGEF is a RhoG-specific bird-derived acid-exchange enzyme, we then examined whether the SGEF enhances the stability of the EGFR protein in a manner that depends on the activity of the avionic acid-exchange enzyme. Sex. The SGEF, which is missing the activity of the avionic acid-exchange enzyme, is still able to inhibit the degradation of the EGFR protein, while finding that the sustained-activated RhoG is not capable of turning on the promotion of the degradation of the EGFR protein by the low SGEF These results indicate that the SGEF enhances the stability of the EGFR protein in a non-dependent manner with the activity of the avionic acid-exchange enzyme. Sex. The degradation of EGFR in the cells is subject to multiple regulation, and after the combination of EGFR with the ligand, dimerization and autophosphorylation, and the EGFR recruitment E3 ubiquitin enzyme Cbl, which phosphorylate, cause the EGFR to occur. Ubiquitin. The ubiquitinated EGFR occurs endocytosis under the mediation of Epsilon-15 and epsin-1. The endocytosis of the EGFR enters the early inner body and then enters the late inner body under the mediation of the ESCRT complex, and finally, in the lysosome, Biodegradation. We then tested which link the SGEF was involved in the reduction of the EGFR protein We found that the overexpression of SGEF did not affect the ubiquitination of EGFR, endocytosis, and the entry of early endosomes, but rather the inhibition of EGFR from early endosomes to late During the period, we found that the knockdown of SGEF in prostate cancer cells did not affect EGFR's entry into the early inner body, but also promoted the entry of EGFR from the early inner body to the night During the period, these results indicate that the SGEF is the inhibition of the EGFR protein by inhibiting the entry of EGFR from the early internal body into the late inner body The high expression of EGFR is usually closely related to the abnormal activation of the EGFR signaling pathway, and we then detected whether the SGEF is capable of regulating the EGF R signal pathway. We found that the knockdown of SGEF significantly inhibited the EGF-induced EGFR and downstream protein AK T phosphorylation. Because the EGFR/ AKT signal pathway plays an important role in the migration of prostate cancer cells, the effect of SGEF on the cell migration of prostate cancer is detected by a scratch test, and the results show that the knockdown of SGEF significantly inhibited the induction of EGF. Cell migration. The EGFR/ ERK1/2 signaling pathway is in a variety of cell life activities It plays an important role. Since we have found that the SGEF is capable of enhancing the stability of the EGFR protein, we then tested the SGEF for EGFR/ ERK1/2 We have found that the expression of SGEF can significantly enhance the phosphorylation of ERK1/2 induced by EGF, while the knockdown of SGEF can significantly inhibit the phosphorylation of ERK1/2 induced by EGF, indicating that the SGEF can enhance the EGFR/ ERK. 1/2 signaling pathway. We then examined whether the SGEF enhanced the EGFR/ ERK1/2 signaling pathway in a RhoG-dependent manner, and we found that the expression of the knockdown RhoG does not inhibit the enhancement of the SGEF to the EGFR/ ERK1/2 signaling pathway, suggesting that the SGEF can enhance the EGFR/ ERK in a RhoG non-dependent manner. 1/2 signaling pathway. Grb2 is in the process of EGF-induced ERK1/2 activation Because the two ends of Grb2 contain the SH3 domain, and the N-terminal of the SGEF contains the Pro domain, and the SH3 domain and the Pro domain have a classical interaction, it is suggested that the SGEF can enhance the EGFR/ E by interacting with the Grb2. The RK1/2 signaling pathway was verified by the GST-pull down experiment and the immunoprecipitation experiment, and then confirmed that the SH3 domain of Grb2 was mediated by the Pro domain of the SGEF. Interaction between the two. But when we detected the effect of the deletion of the SGEF mutant interacting with the Grb2 on the EGFR/ ERK1/2 signaling pathway, we unexpectedly found that the SGEF mutant not only reduced the enhancement of the EGFR/ ERK1/2 signaling pathway compared to the wild-type SGEF, but further enhanced the EGF-induced ERK1/2 phosphorylation, suggesting that Grb2 may antagonize the EGFR/ ERK1/2 by interacting with the SGEF We found that the expression of Grb2 could inhibit the enhancement of the signaling pathway of the EGFR/ ERK1/2 signaling pathway by the expression of Grb2, while the low Gb2 promoted the enhancement of the EGFR/ ERK1/2 signaling pathway and found that the GGrb2 antagonized the enhancement of the EGFR/ ERK1/2 signaling pathway with Grb2 and SG In conclusion, the study found that the SGEF can be used to inhibit the entry of EGFR from the early inner body into the late inner body. The stability of EGFR protein is enhanced. Because of the high expression of EGFR is the main cause of abnormal activation of EGFR signaling pathway in prostate cancer cells, the control of the stability of the EGFR protein may be the leading role of the SGEF in promoting the stability of EGFR protein. One of the causes of the malignant progression of the adenocarcinoma. At the same time, the SGEF has the potential to inhibit the decline of a variety of growth factor receptors in a similar manner due to the fact that the degradation of the various growth factor receptors is consistent with the EGFR. We found that the SGEF can enhance the activation of the EGF-induced ERK1/2 in the non-dependent manner of RhoG, and the Grb2 can antagonize the EGFR/ ERK1 by interacting with the SGEF. The enhancement of the/2 signaling pathway. As Grb2 has been considered to play a positive regulatory role in the EGFR/ ERK1/2 signaling pathway, GGrb2 can negatively regulate the EGFR/ ERK1/2 signaling pathway in the case of overexpression of the SGEF, revealing that Grb2 is in the EGFR/ ERK1/2 signal
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2511220
[Abstract]:The abnormal activation of the EGFR signaling pathway is closely related to the malignant progression of prostate cancer, the high Gleason classification and the androgen-independent process. The most recent study found that only 8% of the EGFR mutations in the prostate tumor specimens, but up to 31% of the samples, showed high EGFR expression, suggesting that the high expression of EGFR was the main cause of the abnormal activation of the EGFR signaling pathway in prostate cancer. The high expression of EGFR in tumor tissue is mainly two reasons, one is gene amplification, and the other is protein degradation. The study did not find a significant correlation between the high expression of EGFR and the amplification of EGFR gene, suggesting that the inhibition of EGFR protein molecule could be the main cause of the abnormal expression of EGFR in prostate cancer. However, the molecular mechanism of the stability and control of EGFR protein in prostate cancer is not clear, and therefore, it is of great significance to find the molecular mechanism of the stability of the new EGFR protein. SGEF is a potential oncogene in the early stage of prostate cancer, and the clinical study shows that the level of SGEF is gradually increasing with the malignant progression of prostate cancer, and we find that the SGEF can promote the proliferation and survival ability of prostate cancer cells. But the specific molecular mechanism of the role of SGEF in prostate cancer is unclear. Our previous studies have found that the knockdown of SGEF can inhibit the phosphorylation of AKT molecules, and AKT is an important molecule downstream of the EGFR, so we assume that the SGEF is likely to control the EGFR/ AKT signal pathway by modulating the stability of the EGFR protein. We first examined whether the SGEF has an effect on the level of EGFR protein expression in prostate cancer cells, and we have found that the knockdown of SGEF reduces the level of EGFR protein expression in prostate cancer cells C4-2B and DU145, suggesting that the SGEF is indeed capable of regulating the expression of EGFR protein in prostate cancer cells In order to further determine the mechanism of SGEF to regulate the level of EGFR protein expression, we have examined the effect of overexpression of SGEF on the degradation of EGFR protein, and it was found that overexpression of SGEF in 293T cells and COS7 cells could significantly reduce the decrease of EGFR protein. Solutions. Then we tested the effect of knock-on low SGEF on the degradation of EGFR protein in a variety of prostate cancer cells, and we found that the knockdown of the low SGEF in a variety of prostate cancer cells could significantly contribute to the reduction of the EGFR protein. These results indicate that the SGEF can enhance the protein expression of EGFR in prostate cancer cells by enhancing the stability of the EGFR protein Ping. Since the SGEF is a RhoG-specific bird-derived acid-exchange enzyme, we then examined whether the SGEF enhances the stability of the EGFR protein in a manner that depends on the activity of the avionic acid-exchange enzyme. Sex. The SGEF, which is missing the activity of the avionic acid-exchange enzyme, is still able to inhibit the degradation of the EGFR protein, while finding that the sustained-activated RhoG is not capable of turning on the promotion of the degradation of the EGFR protein by the low SGEF These results indicate that the SGEF enhances the stability of the EGFR protein in a non-dependent manner with the activity of the avionic acid-exchange enzyme. Sex. The degradation of EGFR in the cells is subject to multiple regulation, and after the combination of EGFR with the ligand, dimerization and autophosphorylation, and the EGFR recruitment E3 ubiquitin enzyme Cbl, which phosphorylate, cause the EGFR to occur. Ubiquitin. The ubiquitinated EGFR occurs endocytosis under the mediation of Epsilon-15 and epsin-1. The endocytosis of the EGFR enters the early inner body and then enters the late inner body under the mediation of the ESCRT complex, and finally, in the lysosome, Biodegradation. We then tested which link the SGEF was involved in the reduction of the EGFR protein We found that the overexpression of SGEF did not affect the ubiquitination of EGFR, endocytosis, and the entry of early endosomes, but rather the inhibition of EGFR from early endosomes to late During the period, we found that the knockdown of SGEF in prostate cancer cells did not affect EGFR's entry into the early inner body, but also promoted the entry of EGFR from the early inner body to the night During the period, these results indicate that the SGEF is the inhibition of the EGFR protein by inhibiting the entry of EGFR from the early internal body into the late inner body The high expression of EGFR is usually closely related to the abnormal activation of the EGFR signaling pathway, and we then detected whether the SGEF is capable of regulating the EGF R signal pathway. We found that the knockdown of SGEF significantly inhibited the EGF-induced EGFR and downstream protein AK T phosphorylation. Because the EGFR/ AKT signal pathway plays an important role in the migration of prostate cancer cells, the effect of SGEF on the cell migration of prostate cancer is detected by a scratch test, and the results show that the knockdown of SGEF significantly inhibited the induction of EGF. Cell migration. The EGFR/ ERK1/2 signaling pathway is in a variety of cell life activities It plays an important role. Since we have found that the SGEF is capable of enhancing the stability of the EGFR protein, we then tested the SGEF for EGFR/ ERK1/2 We have found that the expression of SGEF can significantly enhance the phosphorylation of ERK1/2 induced by EGF, while the knockdown of SGEF can significantly inhibit the phosphorylation of ERK1/2 induced by EGF, indicating that the SGEF can enhance the EGFR/ ERK. 1/2 signaling pathway. We then examined whether the SGEF enhanced the EGFR/ ERK1/2 signaling pathway in a RhoG-dependent manner, and we found that the expression of the knockdown RhoG does not inhibit the enhancement of the SGEF to the EGFR/ ERK1/2 signaling pathway, suggesting that the SGEF can enhance the EGFR/ ERK in a RhoG non-dependent manner. 1/2 signaling pathway. Grb2 is in the process of EGF-induced ERK1/2 activation Because the two ends of Grb2 contain the SH3 domain, and the N-terminal of the SGEF contains the Pro domain, and the SH3 domain and the Pro domain have a classical interaction, it is suggested that the SGEF can enhance the EGFR/ E by interacting with the Grb2. The RK1/2 signaling pathway was verified by the GST-pull down experiment and the immunoprecipitation experiment, and then confirmed that the SH3 domain of Grb2 was mediated by the Pro domain of the SGEF. Interaction between the two. But when we detected the effect of the deletion of the SGEF mutant interacting with the Grb2 on the EGFR/ ERK1/2 signaling pathway, we unexpectedly found that the SGEF mutant not only reduced the enhancement of the EGFR/ ERK1/2 signaling pathway compared to the wild-type SGEF, but further enhanced the EGF-induced ERK1/2 phosphorylation, suggesting that Grb2 may antagonize the EGFR/ ERK1/2 by interacting with the SGEF We found that the expression of Grb2 could inhibit the enhancement of the signaling pathway of the EGFR/ ERK1/2 signaling pathway by the expression of Grb2, while the low Gb2 promoted the enhancement of the EGFR/ ERK1/2 signaling pathway and found that the GGrb2 antagonized the enhancement of the EGFR/ ERK1/2 signaling pathway with Grb2 and SG In conclusion, the study found that the SGEF can be used to inhibit the entry of EGFR from the early inner body into the late inner body. The stability of EGFR protein is enhanced. Because of the high expression of EGFR is the main cause of abnormal activation of EGFR signaling pathway in prostate cancer cells, the control of the stability of the EGFR protein may be the leading role of the SGEF in promoting the stability of EGFR protein. One of the causes of the malignant progression of the adenocarcinoma. At the same time, the SGEF has the potential to inhibit the decline of a variety of growth factor receptors in a similar manner due to the fact that the degradation of the various growth factor receptors is consistent with the EGFR. We found that the SGEF can enhance the activation of the EGF-induced ERK1/2 in the non-dependent manner of RhoG, and the Grb2 can antagonize the EGFR/ ERK1 by interacting with the SGEF. The enhancement of the/2 signaling pathway. As Grb2 has been considered to play a positive regulatory role in the EGFR/ ERK1/2 signaling pathway, GGrb2 can negatively regulate the EGFR/ ERK1/2 signaling pathway in the case of overexpression of the SGEF, revealing that Grb2 is in the EGFR/ ERK1/2 signal
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 Mikio Hoshino;Dennis W.Wolff;Margaret A.Scofield;Frank J.Dowd;;Upregulation of PIP3-Dependent Rac Exchanger 1(P-Rex1) Promotes Prostate Cancer Metastasis[J];生物物理学报;2009年S1期
本文编号：2511220
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