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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

SUMOyaltion of Shp2 promotes ERK activation by controlling its association with Gab1(A) Cytosolic fractions and membranous fractions extracted from EGF-stimulated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were analyzed by Western blotting with antibodies against HA, IGF-1Rβ (as a membrane protein marker) and GAPDH (as a cytosolic marker). (B) 293T or 293T-shSENP1 cells were co-transfected with HA-Shp2 and Flag-Gab1 plasmids. 24 h after transfection, cells were subjected to serum deprivation for 16 h, followed by the treatment with 100 ng/mL of EGF for 5 min. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C) Lysates from EGF-treated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies against pERK1/2 and other indicated. (D) Lysates from EGF-treated SMMC-7721-shSUMO1 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies indicated. (E) Flag-Gab1 was transiently expressed in 293T cells and GST-Shp2 with pE1E2SUMO1 were transformed into E.coli BL21 (DE3). Two reciprocal pull-down assays of GST-proteins (left panels) and anti-Flag/IP (right panels) were performed, and then immunoblotted. (F) Lysates from 293T cells co-transfected with His-SUMO1 and Flag-Gab1WT or Flag-Gab1SIM1/2mut palsmids were immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody. Lysates were also used as Input for immunoblotting with indicated antibodies (left panels). The consensus amino acid sequences (yellow labeled) of predicted SIM1 and SIM2 of Gab1 were mutated to alanine (right panels).
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Figure 6: SUMOyaltion of Shp2 promotes ERK activation by controlling its association with Gab1(A) Cytosolic fractions and membranous fractions extracted from EGF-stimulated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were analyzed by Western blotting with antibodies against HA, IGF-1Rβ (as a membrane protein marker) and GAPDH (as a cytosolic marker). (B) 293T or 293T-shSENP1 cells were co-transfected with HA-Shp2 and Flag-Gab1 plasmids. 24 h after transfection, cells were subjected to serum deprivation for 16 h, followed by the treatment with 100 ng/mL of EGF for 5 min. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C) Lysates from EGF-treated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies against pERK1/2 and other indicated. (D) Lysates from EGF-treated SMMC-7721-shSUMO1 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies indicated. (E) Flag-Gab1 was transiently expressed in 293T cells and GST-Shp2 with pE1E2SUMO1 were transformed into E.coli BL21 (DE3). Two reciprocal pull-down assays of GST-proteins (left panels) and anti-Flag/IP (right panels) were performed, and then immunoblotted. (F) Lysates from 293T cells co-transfected with His-SUMO1 and Flag-Gab1WT or Flag-Gab1SIM1/2mut palsmids were immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody. Lysates were also used as Input for immunoblotting with indicated antibodies (left panels). The consensus amino acid sequences (yellow labeled) of predicted SIM1 and SIM2 of Gab1 were mutated to alanine (right panels).

Mentions: Our above results have proven that Shp2 SUMOylation is crucial for maintaining the ERK activities, sequentially resulting in cellular transformation and enhanced tumorigenesis both in vitro and in vivo, so next we wanted to explore the underlying mechanism. Our previous studies demonstrate that SUMOylation of PTEN facilitates its association with the membrane and mediates PTEN functions [12], also one study reported that Shp2 membrane-localization is activating on ERK phosphorylation in an EGFR-dependent manner [32]. To determine whether Shp2 SUMOylation indeed influences its membrane association, we conducted a cellular fractionation assay with SMMC-7721-shShp2 cell lines stably re-expressing HA-Shp2WT or HA-Shp2K590R after stimulation with EGF for 5 min, showing that Shp2K590R in the membrane fraction was notably reduced compared to that of Shp2WT (Figure 6A). This result indicates that Shp2 SUMOylation is required for the recruitment of Shp2 to the plasma membrane to regulate RAS/MEK/MAPK signaling pathway.


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)

SUMOyaltion of Shp2 promotes ERK activation by controlling its association with Gab1(A) Cytosolic fractions and membranous fractions extracted from EGF-stimulated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were analyzed by Western blotting with antibodies against HA, IGF-1Rβ (as a membrane protein marker) and GAPDH (as a cytosolic marker). (B) 293T or 293T-shSENP1 cells were co-transfected with HA-Shp2 and Flag-Gab1 plasmids. 24 h after transfection, cells were subjected to serum deprivation for 16 h, followed by the treatment with 100 ng/mL of EGF for 5 min. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C) Lysates from EGF-treated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies against pERK1/2 and other indicated. (D) Lysates from EGF-treated SMMC-7721-shSUMO1 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies indicated. (E) Flag-Gab1 was transiently expressed in 293T cells and GST-Shp2 with pE1E2SUMO1 were transformed into E.coli BL21 (DE3). Two reciprocal pull-down assays of GST-proteins (left panels) and anti-Flag/IP (right panels) were performed, and then immunoblotted. (F) Lysates from 293T cells co-transfected with His-SUMO1 and Flag-Gab1WT or Flag-Gab1SIM1/2mut palsmids were immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody. Lysates were also used as Input for immunoblotting with indicated antibodies (left panels). The consensus amino acid sequences (yellow labeled) of predicted SIM1 and SIM2 of Gab1 were mutated to alanine (right panels).
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Figure 6: SUMOyaltion of Shp2 promotes ERK activation by controlling its association with Gab1(A) Cytosolic fractions and membranous fractions extracted from EGF-stimulated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were analyzed by Western blotting with antibodies against HA, IGF-1Rβ (as a membrane protein marker) and GAPDH (as a cytosolic marker). (B) 293T or 293T-shSENP1 cells were co-transfected with HA-Shp2 and Flag-Gab1 plasmids. 24 h after transfection, cells were subjected to serum deprivation for 16 h, followed by the treatment with 100 ng/mL of EGF for 5 min. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C) Lysates from EGF-treated SMMC-7721-shShp2 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies against pERK1/2 and other indicated. (D) Lysates from EGF-treated SMMC-7721-shSUMO1 cells stably re-expressing hShp2WT or hShp2K590R were immunoprecipitated with anti-Gab1 antibody and then immunoblotted with anti-HA antibody. Lysates were also used as Input for immunoblotting with antibodies indicated. (E) Flag-Gab1 was transiently expressed in 293T cells and GST-Shp2 with pE1E2SUMO1 were transformed into E.coli BL21 (DE3). Two reciprocal pull-down assays of GST-proteins (left panels) and anti-Flag/IP (right panels) were performed, and then immunoblotted. (F) Lysates from 293T cells co-transfected with His-SUMO1 and Flag-Gab1WT or Flag-Gab1SIM1/2mut palsmids were immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody. Lysates were also used as Input for immunoblotting with indicated antibodies (left panels). The consensus amino acid sequences (yellow labeled) of predicted SIM1 and SIM2 of Gab1 were mutated to alanine (right panels).
Mentions: Our above results have proven that Shp2 SUMOylation is crucial for maintaining the ERK activities, sequentially resulting in cellular transformation and enhanced tumorigenesis both in vitro and in vivo, so next we wanted to explore the underlying mechanism. Our previous studies demonstrate that SUMOylation of PTEN facilitates its association with the membrane and mediates PTEN functions [12], also one study reported that Shp2 membrane-localization is activating on ERK phosphorylation in an EGFR-dependent manner [32]. To determine whether Shp2 SUMOylation indeed influences its membrane association, we conducted a cellular fractionation assay with SMMC-7721-shShp2 cell lines stably re-expressing HA-Shp2WT or HA-Shp2K590R after stimulation with EGF for 5 min, showing that Shp2K590R in the membrane fraction was notably reduced compared to that of Shp2WT (Figure 6A). This result indicates that Shp2 SUMOylation is required for the recruitment of Shp2 to the plasma membrane to regulate RAS/MEK/MAPK signaling pathway.

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