SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1.
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.
Yingying
Y
Qu
Qin
Q
Chen
Xueping
X
Lai
Changhong
C
Zhu
Cheng
C
Chen
Xian
X
Zhao
Rong
R
Deng
Ming
M
Xu
Haihua
H
Yuan
Yanli
Y
Wang
Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. Jianxiu.Yu@gmail.com.
Jianxiu
J
Yu
Jian
J
Huang
D000595:Amino Acid Sequence; D000818:Animals; D063646:Carcinogenesis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics* / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020837:SOS1 Protein / Q000235:genetics* / Q000378:metabolism; D058207:Sumoylation; D023041:Xenograft Model Antitumor Assays; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D034741:RNA, Small Interfering; D020837:SOS1 Protein; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins