A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.

RPTPα phosphatase activity is allosterically regulated by the membrane-distal catalytic domain.

Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.