Solution structure of SHIP2 SH2 domain and its interaction with a phosphotyrosine peptide from c-MET

Abstract

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.

Structural Insights into the Binding Propensity of Human SHIP2 SH2 to Oncogenic CagA Isoforms from Helicobacter pylori

“…Further, alanine mutation-based analysis was performed to identify key SHIP2-SH2 residues for binding to EPIYA-C and EPIYA-D. Five residues in the pY-pocket, including R28, S49, E50, S51 and R70, and two residues in the electroneutral pocket, including S84 and Q107, were selected for mutagenesis, as referred to in the NMR titration data and the residues selected in our previous studies [ 13 , 21 ]. The binding affinities of these mutants for 13-mer CagA EPIYA-C and EPIYA-D phosphopeptides were determined by FP assay ( Figure 2 E).…”

“…SHIP2 is a multi-domain protein with the 5-phosphatase domain in the middle and several regulatory domains including SH2 and PH-R located in the N-terminal part of the protein, and C2, proline-rich, and the sterile alpha motif (SAM) located in the C-terminal part [ 11 , 12 , 13 , 14 , 15 ]. The SHIP2 SH2 domain (hereafter represented by SHIP2-SH2) can mediate the interaction of SHIP2 with a variety of proteins, such as p130cas [ 16 ], hepatocyte growth factor receptor (HGFR/c-MET) [ 17 ] and immunoreceptors [ 18 , 19 ], through binding to the phosphotyrosine (pY)-containing motifs in these proteins, so as to recruit SHIP2 to the corresponding pY-dependent signaling pathways.…”

“…SHIP2-SH2 adopts a canonical SH2 structure consisting of six β-strands (βA to βF) flanked by two α-helices (αA and αB). A positively charged pY-binding pocket formed by αA, βB, βC, βD and the BC-loop (between βB and βC) can be found in the structure [ 13 ]. Although the pY-motifs in the native interaction partners of SHIP2-SH2 identified to date show low sequence similarity, a common characteristic can be found in that they all have aliphatic residues at the pY+3 positions [ 17 , 18 , 19 , 20 ].…”

“…In recent years, and using NMR methods, we investigated the interaction of SHIP2-SH2 with Tyr-phosphorylated c-MET and FcgR2B, which mediate hepatocyte growth factor (HGF) and immunity signaling, respectively [ 13 , 21 ]. In this study, we further studied the interaction of SHIP2-SH2 with four types of Tyr-phosphorylated EPIYA motifs.…”

<p>ARQ-197 enhances the antitumor effect of sorafenib in hepatocellular carcinoma cells via decelerating its intracellular clearance</p>

“…[19][20][21] On HCC cells, HGF binds and activates c-MET, further inducing the transduction of c-MET downstream pathways, such as Raf/MEK/ERK and AKT/ETS-1 signaling pathways, and leads to the proliferation, migration, and invasion of HCC cells. [22][23][24] Thus, based on the roles of c-MET in many such features of tumor cells, we hypothesized that it may participate in multidrug resistance and the clearance of antitumor agents. In this study, we used ARQ-197 (also named Tivantinib), a highly selective and non-ATP competitive inhibitor of c-MET for HCC cell treatment, and found that it inhibited the activation of c-MET, promoted apoptosis, and inhibited proliferation and metastasis of HCC cells.…”