ITIM/ITSM Domains Of PTPs For PD-1 And PZR Dephosphorylation

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms.This process is regulated by protein tyrosine kinases(PTKs)and protein tyrosinase(PTPs).Protein tyrosine phosphatase plays an important role in cell signal transduction pathway and is a large family of signal enzymes.Its function is removing the phosphate group from the phosphoserine residue of the target protein and thus changes the function of the target protein.The myelin P0 protein-related protein(PZR)is a member of a transmembrane immunoglobulin superfamily and is a specific binding protein and substrate for the tyrosine phosphatase SHP-2.PZR contains two intracellular immunosuppression motifs(ITIMs)that have tyrosine phosphorylation sites.By recruiting SHP-2 to complete downstream signaling,PZR plays an important role in regulating cell cycle and migration.Programmed Death 1(PD-1)is also a transmembrane immunoglobulin with an ITIM and an immunoreceptor tyrosine switch motif(ITSM)in the intracellular region,which is considered as a key functional domain.The tyrosine residue in ITSM can be phosphorylated and recruit intracellular phosphatase,triggering the dephosphorylation of antigen-antibody complexes.Studies have shown that after ligand PD-L1 binds to PD-1,SHP-2 is recruited to the cell membrane through PD-I intracellular ITIM/ITSM,negatively regulating downstream signal transduction pathways,leading to immune-related diseases.Although there is a certain understanding of the process of recruiting phosphatases in ITIM/ITSM,we still know very little about the regulation of phosphorylation of ITIM/ITSM itself.It was found that the Src kinase family catalyzes the phosphorylation of tyrosine residues in ITIM/ITSM,thereby recruiting SHP-2 and causing the dephosphorylation of downstream protein kinases and inhibiting the activation of downstream AKT,ERK,and other signaling pathways.However,the dephosphorylation of ITIM/ITSM remains unknown.Therefore,elucidating the dephosphorylation regulation process of ITIM/ITSM has far-reaching significance in revealing the pathogenesis of related diseases.At the present stage,the most commonly used methods for in vitro detection of protein phospholipids are still antibody detections,but they have certain limitations and cannot accurately study the method,degree and state of dephosphorylation of proteins.In this paper,we first expressed and purified several common non-receptor PTPs,and simultaneously synthesized phosphorylated peptides of ITIM/ITSM domain containing PD-1 and PZR,and assisted laser desorption flight through MALDI-TOF matrix.By means of time-mass spectrometry,the dephosphorylation of phosphorylated ITIM/ITSM by PTPs was investigated in vitro.Experimental results indicate that PTPs are selective for the dephosphorylation of phosphorylated ITIM/ITSM,revealing the substrate specificity of PTPs,which is used to elucidate the phosphorylation regulation mechanism of ITIM/ITSM domain-containing signaling molecules and to targeted drug development,has great theoretical significance.Compared with traditional experimental methods,the mass spectrometry method we use can more quickly,more effectively,and more intuitively understand the dephosphorylation of ITIM/ITSM and enrich the means for studying the activity and biological function of PTPs.