Structural Mechanisms of PD-1 – SHP2 Signaling
Introduction: Colorectal cancer (CRC) is the 3rd most common cancer globally, and the 4th most common cause of cancer mortality1. Inflammatory bowel disease (IBD) ranks as a major predisposing factor for the development of CRC, termed colitis-associated colorectal cancer (CAC) when caused by inflammatory etiologies2. Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) is a signaling molecule known to play a role in the attenuation of immune responses against various malignancies, including CRC. Recent studies indicate that SHP2-deficient mice with CAC experience regression of colorectal cancer symptoms, at the cost of exacerbation of colitis symptoms3. These results, along with the known role of SHP2 in the PD-1 (programmed death protein-1) immunosuppression, indicates that understanding the structural basis for PD1 – SHP2 signaling axis can provide value in treating CRC4. Methods: X-ray crystallography determined the precise mode of PD – 1 ITSM (immune receptor tyrosine-based switch motif) and ITIM (immune receptor tyrosine-based inhibitory motif) binding to SHP2. Subsequently, an activation assay of WT SHP2 using ITIM, ITSM, and ITIM – ITSM peptides chemically “linked” via [dPEG4]2 determined the relative roles of each motif in activating SHP2. Finally, PD-L1-(+) Raji B-cells were incubated with 3 genotypically distinct variants of Jurkat T-cells (WT PD-1, ITIM knockout, and ITSM knockout), with use of SDS-PAGE and immunoblotting to reveal the functional significance of ITIM and ITSM4. Results: X-ray crystallography reveals that the PD-1 ITIM and ITSM preferentially bind to the N-terminal SH2 and C-terminal SH2 domains of SHP2, respectively, relieving the N-SH2 domain autoinhibition of SHP2. The activity assay revealed that ITIM “linked” to ITSM with [dPEG4]2 exhibited superior activation of SHP2 than the individual ITIM and ITSM peptides, indicating that ITIM and ITSM play cooperative roles in the activation of SHP2. Finally, immunoblotting of PD-L1-(+) Raji B-cells and Jurkat T-cells lysates revealed that ITSM knockout Jurkat T-cells cannot activate SHP2, although ITIM knockout T-cells can still bind to SHP2, indicating that ITSM plays indispensible roles in SHP2 activation4. Conclusions: The molecular mechanism for PD-1 activation by SHP2, and, by extension, PD-1-mediated immunosuppression, requires binding by the ITSM motif. However, binding of both the ITIM and ITSM motifs leads to superior activation of SHP2 compared to binding of individual ITIM and ITSM motifs. This indicates the possibility of 2 “modes” of SHP2 activation by PD-1: “strong” activation, which requires simultaneous binding of ITSM and ITIM, and “moderate” activation, which only requires ITSM4.
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