INPP5K limits PtdIns (4,5)P2 accumulation in CD19 microclusters and restrains CD19 co-receptor membrane proximity and BCR-proximal signaling in B cells.

CD19 is a key B-cell co-receptor that amplifies B-cell receptor (BCR) signaling and tunes B-cell activation thresholds. How the conformation of the CD19 cytoplasmic domain is regulated to restrain basal and BCR-induced signaling remains incompletely understood. We investigated whether the phosphoinositide 5-phosphatase INPP5K controls CD19 conformation and CD19-amplified BCR-proximal signaling through local regulation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. A B-cell-specific Inpp5k knock-down (KD) mouse model was generated using CD21-Cre-mediated recombination. CD19-phospholipid interactions were assessed by tryptophan fluorescence with synthetic CD19 peptides and lipid bicelles. CD19 cytoplasmic domain proximity to the plasma membrane was measured by indirect FRET. PtdIns(4,5)P₂ distribution within CD19 microclusters, BCR-proximal signaling, and microcluster dynamics were analyzed by Airyscan microscopy and phospho-flow cytometry. Peripheral B-cell subsets and serum immunoglobulins were quantified by flow cytometry and ELISA. The CD19 juxtamembrane cytoplasmic domain contains a conserved polybasic region that interacts with acidic phospholipids, including PtdIns(4,5)P2. INPP5K KD B cells displayed increased PtdIns(4,5)P2 density within CD19 microclusters and a constitutively membrane-proximal CD19 cytoplasmic domain in resting transitional T1/T2 B cells. This was associated with elevated basal phosphorylation of CD19^Y531^, BTK^Y223^ and PLCγ2^Y759^, with preserved inducibility upon BCR engagement, whereas ITAM-proximal components (CD79A, LYN, SYK) were unchanged or reduced. INPP5K deficiency also increased basal CD19 and BCR microcluster formation, impaired CD19-BCR colocalization after stimulation, and altered ezrin phosphorylation and actin organization. These changes coincided with accumulation of T1 B cells, reduced T2, follicular and marginal-zone B cells, and decreased serum IgM and IgG. INPP5K limits local PtdIns(4,5)P2 accumulation in CD19 microclusters, thereby restraining membrane association of the CD19 cytoplasmic domain and basal CD19-amplified BCR-proximal signaling. Loss of INPP5K skews the spatial organization and balance of BCR-proximal signaling, with downstream consequences for B-cell microcluster dynamics, peripheral differentiation and humoral output.