Cytoskeletal Protein 4.1R Inhibits BCR-Mediated B-Cell Activation by Restraining AKT1 Phosphorylation.

B-cell receptor (BCR) is indispensable for B-cell responses, and its signaling relies on the rearrangement of cytoskeletal proteins. Cytoskeletal protein 4.1R has been previously implicated in the regulation of immune function. However, the specific role of 4.1R in BCR-mediated B-cell activation remains unknown. Here, we performed single-cell RNA (scRNA) sequencing on splenic B cells isolated from wild-type (WT) and 4.1R-knockout (4.1R-KO) mice to systematically characterize the functional contribution of 4.1R to B-cell biology. Transcriptomic analyses suggested a critical role for 4.1R in modulating BCR signaling. Ex vivo stimulation of primary B cells with anti-IgM demonstrated that 4.1R-KO B cells exhibited marked overactivation, hyperproliferation, and enhanced antibody secretion. Furthermore, unbiased phosphoproteomic profiling, identified sustained AKT1 phosphorylation as a key feature in 4.1R-KO B cells. Subsequent functional validation confirmed that 4.1R regulates BCR signaling by constraining AKT1 activation. Mechanistically, 4.1R rapidly colocalized with the coreceptor CD19 at the plasma membrane upon BCR engagement, and co-immunoprecipitation confirmed their physical interaction. Loss of 4.1R disrupted this interaction and resulted in sustained and amplified AKT1 phosphorylation (but not AKT2) in stimulated B cells. Collectively, our findings identify 4.1R as a novel negative regulator of BCR signaling that interacts with CD19 to constrain AKT1 activation.