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

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.