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. 2014 Apr 29:13:95.
doi: 10.1186/1476-4598-13-95.

SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1

Yingying Qu, Qin Chen, Xueping Lai, Changhong Zhu, Cheng Chen, Xian Zhao, Rong Deng, Ming Xu, Haihua Yuan, Yanli Wang, Jianxiu Yu  1 , Jian Huang
Affiliations

SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1

Yingying Qu et al. Mol Cancer. .

Abstract

Background: Grb2 (Growth factor receptor-bound protein 2) is a key adaptor protein in maintaining the ERK activity via linking Sos1 (Son of sevenless homolog 1) or other proteins to activated RTKs, such as EGFR. Currently, little knowledge is available concerning the post-translational modification (PTM) of Grb2 except for its phosphorylation. Since emerging evidences have highlighted the importance of SUMOylation (Small ubiquitin-related modifier), a reversible PTM, in modulating protein functions, we wondered if Grb2 could be SUMOylated and thereby influences its functions especially involved in the Ras/MEK/ERK pathway.

Methods: SUMOylation of Grb2 was analyzed with the in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown and the in vitro E.coli-based SUMOylation assay. To test the ERK activity and cell transformation, the murine fibroblast cell line NIH/3T3 and the murine colon cancer cell line CMT-93 were used for the experiments including Grb2 knockdown, ectopic re-expression, cell transformation and migration. Immunoprecipitation (IP) was employed for seeking proteins that interact with SUMO modified Grb2. Xenograft tumor model in mice was conducted to verify that Grb2 SUMOylation regulated tumorigenesis in vivo.

Results: Grb2 can be SUMOylated by SUMO1 at lysine 56 (K56), which is located in the linker region between the N-terminal SH3 domain and the SH2 domain. Knockdown of Grb2 reduced the ERK activity and suppressed cell motility and tumorigenesis in vitro and in vivo, which were all rescued by stable ectopic re-expression of wild-type Grb2 but not the mutant Grb2K56R. Furthermore, Grb2 SUMOylation at K56 increased the formation of Grb2-Sos1 complex, which sequentially leads to the activation of Ras/MEK/MAPK pathway.

Conclusions: Our results provide evidences that Grb2 is SUMOylated in vivo and this modification enhances ERK activities via increasing the formation of Grb2-Sos1 complex, and may consequently promote cell motility, transformation and tumorigenesis.

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Figures

Figure 1
Figure 1
Grb2 is SUMOylated in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Grb2 along with or without His-SUMO1 and HA-Ubc9. SUMOylated proteins were purified from cell lysates using Ni2+-NTA affinity pulldown (Ni-pulldown) and SUMOylated Grb2 was detected by immunoblotting for the Flag tag. (B) His-SUMO1-conjugated proteins were purified by Ni-pulldown from HEK293T cells expressing Flag-Grb2, His-SUMO1, HA-Ubc9 or SENP1 and probed for Grb2. *indicates endogenous SUMOylated Grb2. (C) HEK293T cells were co-transfected with HA-SUMO1 and Flag-Ubc9. Lysates were immunoprecipitated with anti-HA(m) antibody or mouse IgG (as a control), and then immunoblotted with anti-Grb2 antibody. (D) The pE1E2S1 plasmid was co-transformed with pGEX4T1 (GST) or pGEX4T1-Grb2 (GST-Grb2) into E.coli BL21(DE3). Proteins were purified with GST agarose beads followed by Western blotting analysis.
Figure 2
Figure 2
K56 is a critical site for Grb2 SUMOylation. (A) HEK293T cells were co-transfected with Flag-Grb2 wild-type or mutants along with or without His-SUMO1 and HA-Ubc9. The in vivo SUMOylation assay using Ni2+-NTA beads was conducted as described in Methods. (B) Amino acid sequence alignment of Grb2 partial sequences located between N-SH3 and SH2 domains from different species. The conserved lysine in the linker region (yellow) has been underlined. (C) HEK293T cells were transfected with Flag-Grb2WT or Flag-Grb2K56R plasmid along with the increased amount of His-SUMO1 and HA-Ubc9 plasmids. The levels of SUMOylation were determined as described in Methods. (D) The plasmid GST-Grb2 or GST-Grb2K56R was co-transformed with pE1E2S1 plasmid into BL21(DE3). Western blotting was conducted after GST pulldown.
Figure 3
Figure 3
SUMOylation of Grb2 at K56 promotes cellular transformation. (A) Comparable expression levels of Grb2WT and Grb2K56R in stable NIH/3T3 cell lines generated by polyclonal lentiviral infections were determined by Western blotting. (B) Stable NIH/3T3 cell lines were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 5 × 104 cells/well and layered onto the base. The photographs were taken 3 weeks later. (C) Vasculogenic mimicry assay for stable NIH/3T3 cell lines. Each cell line at a density of 1 × 104 was plated in each well which was coated with matrigel and photographs were taken with microscope 20 hours later. (D) Stable NIH/3T3 cell lines at 60% confluence were stimulated with 5 μg/ml of Doxycycline (Doxy) for 24 or 36 hours. The ERK activities were determined by Western blotting. (E) Densitometric readings were obtained with ImageJ V1.45 software, and the signals of phosphorylated Erk were normalized to that of total Erk. Data are representative of three independent experiments.
Figure 4
Figure 4
SUMOylation of Grb2 promotes migration and tumorigenesis by upregulation of the ERK activities. (A-B) Endogenous Grb2 in the murine fibroblast cell line NIH/3T3 (A) and the murine colon cancer cell line CMT-93 (B) was knockdown by a short hairpin RNA targeting Grb2 3′UTR (shGrb2) by using the lentiviral vector pLKO.1 system. Grb2WT and Grb2K56R were reintroduced respectively into these stable Grb2-silencing NIH/3T3 and CMT-93 cells by the lentiviral system. (C) The ERK activities in stable NIH/3T3 cell lines treated with 5 μg/ml of Doxycycline for 24 or 48 hours were determined by Western blotting. (D) Serum-starved stable CMT-93 cell lines were stimulated with 100 ng/ml of EGF for 5 or 10 minutes and the ERK activities were determined by Western blotting. (E-F) Cell motility was determined by wound healing assay in μ-Dish for NIH/3T3 (E) and CMT-93 (F) stable cell lines. (G) The kinetic information about the migration of CMT-93 stable cell lines was recorded using the x CELLigence RTCA-DP system. (H) Stable CMT-93 cell lines were seeded in 2 ml of medium containing 20% FBS with 0.35% agar at 5 × 103 cells/well and layered onto the base. The photographs were taken 2 weeks later. Images are representative of three independent experiments. (I) Stable CMT-93 cell lines (2 × 106) were injected subcutaneously into male BALB/c nude mice (n = 5) individually. The sizes of tumors were measured at day 14, 17 and 20 after injection and the tumors were weighed.
Figure 5
Figure 5
SUMOylation of Grb2 increases its binding with Sos1. (A-B) HEK293T cells were co-transfected with the indicated plasmids. 24 hours later, cells were subjected to serum deprivation for 20 hours, followed the treatments with or without 100 ng/ml of EGF (A) or 10% serum (B) for the indicated time. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C-D) HEK293T cells were co-transfected with the indicated plasmids and harvested after 48 hours. Cell lysates were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. Densitometric readings were obtained with ImageJ V1.45 software. (E-F) Sos1 was not SUMOylated. HEK293T cells were co-transfected with the indicated plasmids and analyzed for SUMOylation by Ni2+-NTA pulldown (E) or immunoprecipitation (F). SUMOylated Grb2 was used as a positive control of SUMOylation, and mouse IgG as a negative control in immunoprecipitation.
Figure 6
Figure 6
A schematic model of Grb2 SUMOylation-regulated ERK pathway. SUMOylation of Grb2 at K56 enhances the ERK activity via recruiting more Sos1 to form Grb2-Sos1 complex, and consequently promoting cell migration and tumorigenesis. Dashed line----Weak; Solid line----Strong.
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References

    1. Hay RT. SUMO: a history of modification. Mol Cell. 2005;18(1):1–12. doi: 10.1016/j.molcel.2005.03.012. - DOI - PubMed
    1. Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev. 2004;18(17):2046–2059. doi: 10.1101/gad.1214604. - DOI - PubMed
    1. Muller S, Hoege C, Pyrowolakis G, Jentsch S. SUMO, ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol. 2001;2(3):202–210. doi: 10.1038/35056591. - DOI - PubMed
    1. Hayashi T, Seki M, Maeda D, Wang W, Kawabe Y, Seki T, Saitoh H, Fukagawa T, Yagi H, Enomoto T. Ubc9 is essential for viability of higher eukaryotic cells. Exp Cell Res. 2002;280(2):212–221. doi: 10.1006/excr.2002.5634. - DOI - PubMed
    1. Verger A, Perdomo J, Crossley M. Modification with SUMO: a role in transcriptional regulation. EMBO reports. 2003;4(2):137–142. doi: 10.1038/sj.embor.embor738. - DOI - PMC - PubMed

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