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.

A dominant function of LynB kinase in preventing autoimmunity.

Here, we report that the LynB splice variant of the Src-family kinase Lyn exerts a dominant immunosuppressive function in vivo, whereas the LynA isoform is uniquely required to restrain autoimmunity in female mice. We used CRISPR-Cas9 gene editing to constrain lyn splicing and expression, generating single-isoform LynA knockout (LynAKO) or LynBKO mice. Autoimmune disease in total LynKO mice is characterized by production of antinuclear antibodies, glomerulonephritis, impaired B cell development, and overabundance of activated B cells and proinflammatory myeloid cells. Expression of LynA or LynB alone uncoupled the developmental phenotype from the autoimmune disease: B cell transitional populations were restored, but myeloid cells and differentiated B cells were dysregulated. These changes were isoform-specific, sexually dimorphic, and distinct from the complete LynKO. Despite the apparent differences in disease etiology and penetrance, loss of either LynA or LynB had the potential to induce severe autoimmune disease with parallels to human systemic lupus erythematosus (SLE).

Regulation of myeloid-cell activation.

Myeloid cells (macrophages, monocytes, dendritic cells, and granulocytes) survey the body for signs of infection and damage and regulate tissue homeostasis, organogenesis, and immunity. They express receptors that initiate the inflammatory response, send signals that alter the vascular and cytokine milieu, and oversee the recruitment, differentiation, and activation of other myeloid and adaptive immune cells. Their activation must therefore be tightly regulated, optimized for maximal innate-immune protection with a minimum of collateral tissue damage or disorganization. In this review we discuss what it means for myeloid cells to become activated, with emphasis on the receptors and signaling molecules important for the recognition of pathogen-associated and damage-associated molecular patterns. We also outline how these signals are regulated by the steric properties of proteins, by adhesive and cytoskeletal interactions, and by negative feedback to keep inflammation in check and support healthy tissue development and homeostasis. Throughout the text we highlight recent publications and reviews and direct readers therein for a comprehensive bibliography.

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.

Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein-protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors. Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near-infrared imaging Alternate Protocol: Visualizing immunoblots using chemiluminescence Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation Support Protocol: Covalent conjugation of antibodies to functionalized beads Basic Protocol 3: Quantifying proteins and post-translational modifications by targeted mass spectrometry.

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.