Membrane contact site resident PTP1B limits superoxide production by suppressing a Syk-Shc1-Phagocyte Oxidase relay

Abstract Phagocytosis is a specialized endocytic process used by macrophages and dendritic cells to engulf particles, which requires coordinated signaling cascades, cytoskeletal remodeling, and assembly of antimicrobial machinery to eliminate pathogens. During Fc γ receptor (FcγR)-mediated phagocytosis, dynamic actin depolymerization at the base of the phagocytic cup creates permissive conditions for endoplasmic reticulum-plasma membrane (ER-PM) membrane contact sites (MCS) to form. We demonstrate that the ER-resident protein tyrosine phosphatase PTP1B localizes to newly formed or expanded ER-PM MCS during phagocytosis and dephosphorylates Syk. Using TIRF microscopy with MCS residents, including MAPPER, STIM1, and E-Syts, we show that actin clearance allows ER proteins to approach the plasma membrane. PTP1B colocalizes with FcγRs in actin-cleared zones and physically interacts with Syk, a critical mediator of phagocytic signaling. Loss of PTP1B led to sustained Syk hyperphosphorylation without affecting phagocytosis. However, the PTP1B-deficient cells showed a ≍3-fold increase in NADPH oxidase 2 (NOX2)-mediated superoxide production. Using unbiased proteomics, we identified the adapter protein Shc1 as a critical intermediate linking Syk phosphorylation to NOX2 activation. Shc1 phosphorylation during phagocytosis is dependent on Src family kinases and Syk, while genetic ablation of SHC1 reduced superoxide production by ≍40%. Proximity ligation assays reveal enhanced Shc1-p47phox interactions in PTP1B-deficient cells during phagocytosis. These findings establish an SFK-Syk-Shc1-NOX2 signaling axis that PTP1B negatively regulates at MCS between the ER and the forming phagosome, providing new mechanistic insights into antimicrobial responses during phagocytosis. Competing Interest Statement The authors have declared no competing interest. Footnotes Addition of an author after locating new contact/email Minor change to Mass Spectrometry methods Clarified legend for Figure 7.