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

Chuling Zhuang; Shen Yang; Carlos G Gonzalez; Richard I Ainsworth; Sheng Li; Masumi Takayama Kobayashi; Igor Wierzbicki; Leigh-Ana M Rossitto; Yutao Wen; Wolfgang Peti; Stephanie M Stanford; David J Gonzalez; Ramachandran Murali; Eugenio Santelli; Nunzio Bottini

doi:10.1016/j.jbc.2024.107393

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

J Biol Chem. 2024 Jun;300(6):107393. doi: 10.1016/j.jbc.2024.107393. Epub 2024 May 21.

Authors

Affiliations

¹ Department of Medicine, Altman Clinical and Translational Research Institute, University of California, San Diego, California, USA.
² Department of Medicine, Altman Clinical and Translational Research Institute, University of California, San Diego, California, USA; Department of Medicine, Kao Autoimmunity Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.
³ Department of Pharmacology, University of California, San Diego, California, USA; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA.
⁴ Department of Medicine, Kao Autoimmunity Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.
⁵ Department of Medicine, University of California, San Diego, California, USA.
⁶ Department of Molecular Biology and Biophysics, University of Connecticut Health, Farmington, Connecticut, USA.
⁷ Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, USA; Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.
⁸ Department of Medicine, Altman Clinical and Translational Research Institute, University of California, San Diego, California, USA; Department of Medicine, Kao Autoimmunity Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. Electronic address: Nunzio.Bottini@cshs.org.

Abstract

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.

Keywords: PTPN22; T cell receptor; allosteric regulation; autoimmunity; catalysis; intrinsically disordered region; phosphoproteomics; phosphorylation; transcriptomics; tyrosine phosphatase.

MeSH terms

Gain of Function Mutation
HEK293 Cells
Humans
Jurkat Cells
Phosphorylation
Protein Tyrosine Phosphatase, Non-Receptor Type 22* / genetics
Protein Tyrosine Phosphatase, Non-Receptor Type 22* / metabolism
Receptors, Antigen, T-Cell* / immunology
Receptors, Antigen, T-Cell* / metabolism
Signal Transduction*
T-Lymphocytes / immunology
T-Lymphocytes / metabolism

Substances

Receptors, Antigen, T-Cell
Protein Tyrosine Phosphatase, Non-Receptor Type 22
PTPN22 protein, human