| The protein tyrosine phosphatase 2(SHP2)containing Src homology domain 2is encoded by the PTPN11 gene and is a key allosteric phosphatase in many signal pathways.The activating mutation of SHP2 can cause AML,gastric cancer,breast cancer and other cancers.Therefore,PTPN11 has now become a recognized proto-oncogene and the only proto-oncogene in the PTP family.The immunoreceptor tyrosine inhibitory motif(ITIM)is a protein sequence with the structure of L/I/V/SXYXXL/V(where X represents any amino acid residue),which usually exists in the membrane that exerts an immunosuppressive effect.The immunoreceptor tyrosine-based switch motif(ITSM)is a motif containing the TXYXXV/I structure.It is often adjacent to ITIM and regulates the activity of phosphatase together with ITIM.Studies have shown that phosphorylated ITIM/ITSM can activate SHP2,but the question of how ITIM/ITSM interacts with the two SH2 domains of SHP2 is still unknown.Exploring the mechanism of action of ITIM/ITSM and SHP2 will provide a better understanding of how SHP2 is activated and the development of SHP2 inhibitors are of great significance.In order to explore the above problems,we have carried out the following work.(1)It is known that the C-terminal end of SHP2 will affect its activity,so we used the original p RC-CMV-SHP2 plasmid in the laboratory as a template to construct a human SHP2 C-terminal truncated recombinant plasmid p ET28a-SHP2 T,the target protein SHP2 T was obtained by affinity chromatography.(2)Enzymatic characterization of SHP2 T was carried out,and the optimal reaction temperature,optimal pH value and optimal ionic strength of the enzyme were explored.(3)Under different conditions,we used PZR and PD-1's ITIM/ITSM monophosphorylated peptides,diphosphorylated peptides,and monophosphorylated peptides in equimolar mixtures to explore the role of ITIM/ITSM in activating SHP2 mechanism.(4)Construction of recombinant plasmids of the three mutants of human SHP2T: the plasmid in which arginine at position 32 of SHP2 T was mutated to lysine(p ET28a-SHP2T_R32K),the plasmid where arginine at position 138 was mutated to lysine(p ET28a-SHP2T_R138K)and the plasmid(p ET28a-SHP2T_DRK)in which the 32 nd and 138 th arginines were simultaneously mutated to lysine.Three mutants of SHP2 T were obtained by affinity chromatography.(5)The activation effect of ITIM/ITSM on the three mutants was studied,and the key amino acid residues affecting the activation of SHP2 were explored.The results showed that(1)the activation effect of monophosphorylated ITIM/ITSM on SHP2 T is very weak,and the equimolar mixture of the two has very limited activation effect on SHP2,which is comparable to the activation effect of double phosphorylated peptides.can be ignored.(2)Compared with pITIM(N),PD-1's p ITSM(C)and PZR's pITIM(C)have a stronger effect on activating SHP2 T.In addition,compared with the pITIM(N)-pITIM(C)bisphosphorylated peptide of PZR,the pITIM-p ITSM bisphosphorylated peptide of PD-1 has a more significant activation effect on SHP2 T,indicating that the combination of p ITSM and SH2 domain is more conducive to SHP2 T Activation.(3)Regardless of monophosphorylated peptides,ITIM/ITSM equimolar mixtures or double phosphorylated peptides,they have almost no activating effect on SHP2T-R32 K mutants,but have weak activating effects on SHP2T-R138 K mutants,indicating that N-The arginine in the SH2 domain plays a key role in the conformational changes of SHP2.In summary,only ITIM/ITSM double phosphorylated peptides with a specific structure can fully activate SHP2 T.Monophosphorylated ITIM/ITSM or peptides with an equimolar mixture of the two have very weak activation effect on SHP2 T.The arginine residue in the N-SH2 domain of SHP2 is the key to the conformational change of SHP2.This article preliminarily reveals the molecular mechanism of ITIM/ITSM activation of SHP2,which provides an important experimental basis for further research on the activation mechanism of SHP2 and the design of SHP2 small molecule inhibitors. |
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