Membrane contact site resident PTP1B limits superoxide production by suppressing a Syk-Shc1-Phagocyte Oxidase relay
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eLife Assessment
This is an important study showing the interaction of the endoplasmic reticulum (ER)-resident tyrosine phosphatase PTP1B with the developing phagocytic cup in macrophages, and its role in inhibiting microbicidal superoxide production. The authors show convincing evidence that PTP1B interacts with Syk, a plasma membrane tyrosine kinase that plays an essential role in phagocytosis, and that ablation of PTP1B increases superoxide production and Syk phosphorylation without affecting phagocytosis. Further evidence suggests that PTP1B may inhibit a Syk/Shc1/NOX2 axis; however, robust demonstration of the proposed chain of events and of the actual role of ER-plasma membrane contact sites in the PTP1B-dependent downregulation of NOX2 activity will require additional experimental evidence. The integration of advanced imaging methods to study contact site formation with functional assays related to phagocytosis and signaling is inspiring.
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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.
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eLife Assessment
This is an important study showing the interaction of the endoplasmic reticulum (ER)-resident tyrosine phosphatase PTP1B with the developing phagocytic cup in macrophages, and its role in inhibiting microbicidal superoxide production. The authors show convincing evidence that PTP1B interacts with Syk, a plasma membrane tyrosine kinase that plays an essential role in phagocytosis, and that ablation of PTP1B increases superoxide production and Syk phosphorylation without affecting phagocytosis. Further evidence suggests that PTP1B may inhibit a Syk/Shc1/NOX2 axis; however, robust demonstration of the proposed chain of events and of the actual role of ER-plasma membrane contact sites in the PTP1B-dependent downregulation of NOX2 activity will require additional experimental evidence. The integration of advanced imaging …
eLife Assessment
This is an important study showing the interaction of the endoplasmic reticulum (ER)-resident tyrosine phosphatase PTP1B with the developing phagocytic cup in macrophages, and its role in inhibiting microbicidal superoxide production. The authors show convincing evidence that PTP1B interacts with Syk, a plasma membrane tyrosine kinase that plays an essential role in phagocytosis, and that ablation of PTP1B increases superoxide production and Syk phosphorylation without affecting phagocytosis. Further evidence suggests that PTP1B may inhibit a Syk/Shc1/NOX2 axis; however, robust demonstration of the proposed chain of events and of the actual role of ER-plasma membrane contact sites in the PTP1B-dependent downregulation of NOX2 activity will require additional experimental evidence. The integration of advanced imaging methods to study contact site formation with functional assays related to phagocytosis and signaling is inspiring.
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Joint Public Review:
Summary:
This study uses state-of-the-art imaging approaches to show that membrane contact site (MCS) markers and the ER-resident tyrosine phosphatase PTP1B accumulate on phagocytic membranes within actin-devoid zones during frustrated phagocytosis in RAW264.7 macrophages. The authors convincingly show that PTP1B interacts with Syk, an Fcγ receptor-associated tyrosine kinase that plays a critical role in phagocytosis, and that ablation of PTP1B results in hyperphosphorylation of Syk and increased superoxide production, without impacting phagocytic efficiency. Using a phosphoproteomic approach, the authors identify the adaptor protein Shc1 as a strongly phosphorylated protein during stimulation of immunoglobulin receptors by aggregated IgG. In the absence of PTP1B, the authors demonstrate an increased interaction …
Joint Public Review:
Summary:
This study uses state-of-the-art imaging approaches to show that membrane contact site (MCS) markers and the ER-resident tyrosine phosphatase PTP1B accumulate on phagocytic membranes within actin-devoid zones during frustrated phagocytosis in RAW264.7 macrophages. The authors convincingly show that PTP1B interacts with Syk, an Fcγ receptor-associated tyrosine kinase that plays a critical role in phagocytosis, and that ablation of PTP1B results in hyperphosphorylation of Syk and increased superoxide production, without impacting phagocytic efficiency. Using a phosphoproteomic approach, the authors identify the adaptor protein Shc1 as a strongly phosphorylated protein during stimulation of immunoglobulin receptors by aggregated IgG. In the absence of PTP1B, the authors demonstrate an increased interaction between Shc1 and the NADPH oxidase NOX2 subunit p47phox, suggesting that PTP1B controls superoxide production by inhibiting a Syk-Shc1-NOX2 axis.
Strengths:
This is a well-reasoned and cogently developed study that uses contemporary methods, including high-quality TIRF microscopy combined with MAPPER (Membrane-Attached Peripheral ER) or SPLICS (split-GFP-based contact site sensors), to describe how membrane contact site markers and the ER-resident tyrosine phosphatase PTP1B accumulate in the phagocytic cup as cortical actin depolymerizes. The genetic data also convincingly show that PTP1B ablation increases Syk and Shc1 phosphorylation, enhances the Shc1/p47phox interaction, and elevates superoxide production, whereas depletion of Shc1 reduces superoxide levels. Overall, the work outlines an interesting interplay between membrane contact sites, signaling, and the phagocytic machinery of broad interest.
Weaknesses:
While the authors indicate that the PTP1B phosphatase downregulates superoxide production via the Syk-Shc1-NOX2 axis and present a summary model depicting the proposed sequence of events, the supporting data are currently mostly circumstantial. For example, although it is clear that PTP1B depletion increases superoxide production as well as Syk and Shc1 phosphorylation in vivo, there are no data directly demonstrating that the effects of PTP1B depletion on superoxide production require enhanced Syk or Shc1 phosphorylation. Likewise, although PTP1B depletion increases the interaction between Shc1 and p47phox, a soluble component of NOX2, there is no compelling demonstration that superoxide production in PTP1B-depleted cells truly depends on the NOX2 complex or on the Shc1/p47phox interaction.
In addition, while the authors elegantly demonstrate the formation of ER-PM contact sites during frustrated phagocytosis within the actin clearance zone, as well as the localization of the PTP1B phosphatase in the same region, it remains unclear whether the presence of the phosphatase at membrane contact sites is required for its regulatory effect on superoxide production.Finally, it would be interesting to investigate these phenomena in other macrophage cell lines and perhaps also in more physiological contexts than frustrated phagocytosis. This would help evaluate the broader generalizability of the results and conclusions.
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