4.7 Article

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding

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SCIENTIFIC REPORTS
卷 12, 期 1, 页码 -

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NATURE PORTFOLIO
DOI: 10.1038/s41598-022-09589-9

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  1. National Institutes of Health [T32AR007304, R01AR073966, T32DA007097]
  2. University of Minnesota Foundation Equipment Award [E-0918-01]

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Csk is a negative regulator of Src-family kinases (SFKs) through phosphorylation and binding with phosphatases. It can homodimerize and its interactions are important for its regulatory function. Investigating Csk homodimerization and phosphatase interactions may reveal new aspects of SFK regulation.
The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction in cells, however, decreased the negative-regulatory function of Csk. Csk W47A, a substitution previously reported to block PTPN22 binding, had a secondary effect of impairing homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

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