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Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development.

Deng R, Zhao X, Qu Y, Chen C, Zhu C, Zhang H, Yuan H, Jin H, Liu X, Wang Y, Chen Q, Huang J, Yu J - Oncotarget (2015)

Bottom Line: Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K590) in its C-terminus, which is reduced by SUMO1-specific protease SENP1.Furthermore, we find that mutant Shp2(K590R) reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1.In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Shp2, an ubiquitously expressed protein tyrosine phosphatase, is essential for regulation of Ras/ERK signaling pathway and tumorigenesis. Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K590) in its C-terminus, which is reduced by SUMO1-specific protease SENP1. Analysis of wild-type Shp2 and SUMOylation-defective Shp2(K590R) mutant reveals that SUMOylation of Shp2 promotes EGF-stimulated ERK signaling pathway and increases anchorage-independent cell growth and xenografted tumor growth of hepatocellular carcinoma (HCC) cell lines. Furthermore, we find that mutant Shp2(K590R) reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1. More surprisingly, we show that human Shp2 (hShp2) and mouse Shp2 (mShp2) have differential effects on ERK activation as a result of different SUMOylation level, which is due to the event of K590 at hShp2 substituted by R594 at mShp2. In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.

No MeSH data available.


Related in: MedlinePlus

SUMOylation of K590 at Shp2 promotes ERK activation(A) SMMC-7721-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or -truncated form (amino acids 1–587) were starved overnight, and then stimulated by EGF for 5 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2 and GAPDH (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (B) HepG2-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or Shp2K590R-SUMO1 (a fusion construct) were stimulated with EGF for 5 min as before, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (C) Endogenous SUMO1 in SMMC 7721 and HepG2 was knockdown by a short hairpin RNA targeting 3′-UTR of SUMO1 mRNA (shSUMO1) by using lentiviral vector pLKO.1 system, and the ERK1/2 activities were determined. (D) SMMC-7721-shSUMO1 and HepG2-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were serum-starved, and then stimulated with 100 ng/mL of EGF for 5 min, and the ERK activities were determined by Western blotting. (E) Soft agar colony forming assays, HepG2-shSUMO1 and SMMC-7721-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 104 and 2 × 103 cells/well, respectively. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each.
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Figure 4: SUMOylation of K590 at Shp2 promotes ERK activation(A) SMMC-7721-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or -truncated form (amino acids 1–587) were starved overnight, and then stimulated by EGF for 5 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2 and GAPDH (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (B) HepG2-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or Shp2K590R-SUMO1 (a fusion construct) were stimulated with EGF for 5 min as before, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (C) Endogenous SUMO1 in SMMC 7721 and HepG2 was knockdown by a short hairpin RNA targeting 3′-UTR of SUMO1 mRNA (shSUMO1) by using lentiviral vector pLKO.1 system, and the ERK1/2 activities were determined. (D) SMMC-7721-shSUMO1 and HepG2-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were serum-starved, and then stimulated with 100 ng/mL of EGF for 5 min, and the ERK activities were determined by Western blotting. (E) Soft agar colony forming assays, HepG2-shSUMO1 and SMMC-7721-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 104 and 2 × 103 cells/well, respectively. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each.

Mentions: Since human Shp2 protein contains 593 amino acids and K590 is exactly located in the C-terminus of GLMQ587QQK590SFR593, we cut off 6 terminal amino acids Q588QK590SFR593 to get the truncated Shp2WT(1–587), which simulates a form of SUMO-deficient Shp2 like Shp2K590R. Similarly as Shp2K590R, the Shp2WT(1–587) indeed impaired ERK phosphorylation when transfected by the lentiviral system in SMMC-7721-shShp2 cells (Figure 4A). Next, to simulate a form of SUMOylated Shp2, we generated a Shp2K590R-SUMO1 (amino acids 2–96) fusion expression construct as described before [12] and transfect HepG2-shShp2 cells. As expected, the impaired ERK phosphorylations by the point mutation of K590R was rescued by Shp2K590R-SUMO1 fusion (Figure 4B). These data suggest that full activation of ERK probably requires SUMO1 modification of Shp2.


Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development.

Deng R, Zhao X, Qu Y, Chen C, Zhu C, Zhang H, Yuan H, Jin H, Liu X, Wang Y, Chen Q, Huang J, Yu J - Oncotarget (2015)

SUMOylation of K590 at Shp2 promotes ERK activation(A) SMMC-7721-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or -truncated form (amino acids 1–587) were starved overnight, and then stimulated by EGF for 5 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2 and GAPDH (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (B) HepG2-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or Shp2K590R-SUMO1 (a fusion construct) were stimulated with EGF for 5 min as before, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (C) Endogenous SUMO1 in SMMC 7721 and HepG2 was knockdown by a short hairpin RNA targeting 3′-UTR of SUMO1 mRNA (shSUMO1) by using lentiviral vector pLKO.1 system, and the ERK1/2 activities were determined. (D) SMMC-7721-shSUMO1 and HepG2-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were serum-starved, and then stimulated with 100 ng/mL of EGF for 5 min, and the ERK activities were determined by Western blotting. (E) Soft agar colony forming assays, HepG2-shSUMO1 and SMMC-7721-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 104 and 2 × 103 cells/well, respectively. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496222&req=5

Figure 4: SUMOylation of K590 at Shp2 promotes ERK activation(A) SMMC-7721-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or -truncated form (amino acids 1–587) were starved overnight, and then stimulated by EGF for 5 min. Cell lysates were used for immunoblotting analysis of phospho-ERK1/2, ERK1/2 and GAPDH (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (B) HepG2-shShp2 cells stably re-expressing Shp2WT, Shp2K590R or Shp2K590R-SUMO1 (a fusion construct) were stimulated with EGF for 5 min as before, and then the ERK activities were determined by Western blotting (left panels). The data are presented as the mean ± s.d. (n = 3) (right panels). (C) Endogenous SUMO1 in SMMC 7721 and HepG2 was knockdown by a short hairpin RNA targeting 3′-UTR of SUMO1 mRNA (shSUMO1) by using lentiviral vector pLKO.1 system, and the ERK1/2 activities were determined. (D) SMMC-7721-shSUMO1 and HepG2-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were serum-starved, and then stimulated with 100 ng/mL of EGF for 5 min, and the ERK activities were determined by Western blotting. (E) Soft agar colony forming assays, HepG2-shSUMO1 and SMMC-7721-shSUMO1 stably expressing Shp2WT or Shp2K590R cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 1 × 104 and 2 × 103 cells/well, respectively. The photographs were taken 20 days later and the number of colonies was scored. Each value represents the mean ± s.e.m. of three independent experiments with triplicates each.
Mentions: Since human Shp2 protein contains 593 amino acids and K590 is exactly located in the C-terminus of GLMQ587QQK590SFR593, we cut off 6 terminal amino acids Q588QK590SFR593 to get the truncated Shp2WT(1–587), which simulates a form of SUMO-deficient Shp2 like Shp2K590R. Similarly as Shp2K590R, the Shp2WT(1–587) indeed impaired ERK phosphorylation when transfected by the lentiviral system in SMMC-7721-shShp2 cells (Figure 4A). Next, to simulate a form of SUMOylated Shp2, we generated a Shp2K590R-SUMO1 (amino acids 2–96) fusion expression construct as described before [12] and transfect HepG2-shShp2 cells. As expected, the impaired ERK phosphorylations by the point mutation of K590R was rescued by Shp2K590R-SUMO1 fusion (Figure 4B). These data suggest that full activation of ERK probably requires SUMO1 modification of Shp2.

Bottom Line: Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K590) in its C-terminus, which is reduced by SUMO1-specific protease SENP1.Furthermore, we find that mutant Shp2(K590R) reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1.In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Shp2, an ubiquitously expressed protein tyrosine phosphatase, is essential for regulation of Ras/ERK signaling pathway and tumorigenesis. Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K590) in its C-terminus, which is reduced by SUMO1-specific protease SENP1. Analysis of wild-type Shp2 and SUMOylation-defective Shp2(K590R) mutant reveals that SUMOylation of Shp2 promotes EGF-stimulated ERK signaling pathway and increases anchorage-independent cell growth and xenografted tumor growth of hepatocellular carcinoma (HCC) cell lines. Furthermore, we find that mutant Shp2(K590R) reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1. More surprisingly, we show that human Shp2 (hShp2) and mouse Shp2 (mShp2) have differential effects on ERK activation as a result of different SUMOylation level, which is due to the event of K590 at hShp2 substituted by R594 at mShp2. In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.

No MeSH data available.


Related in: MedlinePlus