SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding.

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.

The Src-family Kinase Lyn in Immunoreceptor Signaling.

Effective regulation of immune-cell activation is critical for ensuring that the immune response, and inflammation generated for the purpose of pathogen elimination, are limited in space and time to minimize tissue damage. Autoimmune disease can occur when immunoreceptor signaling is dysregulated, leading to unrestrained inflammation and organ damage. Conversely, tumors can coopt the tissue healing and immunosuppressive functions of hematopoietic cells to promote metastasis and evade therapy. The Src-family kinase Lyn is an essential regulator of immunoreceptor signaling, initiating both proinflammatory and suppressive signaling pathways in myeloid immune cells (eg, neutrophils, dendritic cells, monocytes, macrophages) and in B lymphocytes. Defects in Lyn signaling are implicated in autoimmune disease, but mechanisms by which Lyn, expressed along with a battery of other Src-family kinases, may uniquely direct both positive and negative signaling remain incompletely defined. This review describes our current understanding of the activating and inhibitory contributions of Lyn to immunoreceptor signaling and how these processes contribute to myeloid and B-cell function. We also highlight recent work suggesting that the 2 proteins generated by alternative splicing of lyn, LynA and LynB, differentially regulate both immune and cancer-cell signaling. These principles may also extend to other Lyn-expressing cells, such as neuronal and endocrine cells. Unraveling the common and cell-specific aspects of Lyn function could lead to new approaches to therapeutically target dysregulated pathways in pathologies ranging from autoimmune and neurogenerative disease to cancer.

Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.