A Phosphatase Membrane-Shuttle Enables Feedback Control of B-Cell Signaling

Mechanisms of local kinase recruitment to amplify signaling in lipid rafts have been extensively studied. However, mechanisms of phosphatase membrane-recruitment to terminate local signaling activity remained elusive. Here we identified CD25 (IL2Rα) as a central phosphatase membrane-shuttle and feedback regulator of B-cell receptor (BCR)-signaling in activated and transformed B-cells. This was unexpected because CD25 is known as one of three chains of the IL2-receptor on T- and NK-cells. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells recruits an inhibitory phosphatase complex for feedback control of BCR-signaling and its oncogenic mimics. Reminiscent of genetic phosphatase-ablation, conditional CD25-deletion profoundly depleted early B-cell development followed by dramatic expansion and hyperactivation of mature B-cell subsets and autoimmunity owing to imbalances of BCR-signaling. However, defects in CD25 deficient mice were not replicated in mice that express CD25 but lack expression of the IL2 cytokine, demonstrating IL2-independent functions of CD25 in B-cells. In six clinical cohorts, high expression levels of CD25 predicted poor outcomes for patients with pre-germinal center (GC), in contrast to favorable outcomes for patients with post-GC B-cell malignancies. Consistent with opposite clinical outcome-annotation, genetic CD25-deletion in B-cell precursor leukemia induced rapid cell death but accelerated proliferation in post-GC B-cell lymphomas. Mechanistically, BCR-engagement or oncogenic BCR-signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail at S268. Genetic deletion, in vitro kinase, and interactome studies revealed that PKCδ-dependent CD25-S268 phosphorylation triggered the PKCδ-scaffold RACK1 to recruit inhibitory phosphatases (SHP1, SHIP1) for membrane-translocation and feedback control of BCR-signaling. Deletion of CD25 in human B-cells induced autonomous BCR-signaling and Ca²⁺-oscillations: In the absence of CD25, inhibitory phosphatases were no longer recruited to the cell membrane to control duration and strength of BCR-signaling. Genetic rescue experiments identified membrane-tethered RACK1 and CD25-S268 tail-phosphorylation as central structural elements of this previously unrecognized phosphatase membrane-shuttle. As a result, CRISPR-mediated knock-in of CD25-S268A mutant that abrogates the formation of inhibitory phosphatase complexes caused accelerated proliferation with hyperactivation of BCR-signaling in primary human tonsillar germinal center B-cells.

In conclusion, PKCδ-mediated phosphorylation of CD25 assembles RACK1-dependent inhibitory phosphatase complexes to enable feedback control of BCR-signaling to prevent autoimmunity and malignant transformation.

Marson:Arsenal Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Compensated co-founder; Spotlight Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Compensated co-founder; Survey Genomics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Co-founder; NewLimit: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; PACT Pharma: Current equity holder in private company, Other: Speaking and/or advising fees; 23andMe: Other: Speaking and/or advising fees; Juno Therapeutics: Other: Speaking and/or advising fees, Research Funding; Trizell: Other: Speaking and/or advising fees; Vertex: Other: Speaking and/or advising fees; Merck: Divested equity in a private or publicly-traded company in the past 24 months, Other: Speaking and/or advising fees; Amgen: Other: Speaking and/or advising fees; Genentech: Other: Speaking and/or advising fees; AlphaSights: Other: Speaking and/or advising fees; Rupert Case Management: Other: Speaking and/or advising fees; Bernstein: Other: Speaking and/or advising fees; ALDA: Other: Speaking and/or advising fees; Offline Ventures: Other: Investor, informal advisor; EPIQ: Other: Client; Epinomics: Research Funding; Sanofi: Research Funding; GlaxoSmithKline: Research Funding; Gilead: Research Funding; Anthem: Research Funding.