SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover.

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.

Reduction of disulfide bonds within anti-D results in enhanced Fcgamma receptor blockade.

BACKGROUND: We have investigated whether chemicals known to disrupt disulfide bonds are capable of altering immunoglobulin anti-D structure resulting in an increased efficacy of the chemically modified anti-D to inhibit Fcgamma receptor (FcgammaR)-mediated phagocytosis. If successful, this would provide a rationale to explore this mechanism of enhancing FcgammaR blockade for future use in immunoglobulin therapies for immune cytopenias. STUDY DESIGN AND METHODS: Anti-D that was shown to block 50 percent of the FcgammaR-mediated phagocytosis of opsonized red blood cells (RBCs) using a monocyte monolayer assay (MMA) was combined with two different thiol-containing compounds, dithiothreitol (DTT) or p-toluenesulfonylmethyl mercaptan, with or without treatment with iodoacetamide, and allowed to react. Excess chemical was removed by extensive dialysis. FcgammaR blockade was assessed by MMA with dialyzed, untreated, or chemically treated anti-D using both D+ and D- opsonized target RBCs. Toxicity was determined by fluorescence-activated cell sorting. Aggregates and oligomerization of chemically treated anti-D were examined using gel filtration-high pressure liquid chromatography. RESULTS: Using disulfide-reducing compounds to chemically modify anti-D significantly increases the efficacy of the anti-D to induce an FcgammaR blockade and decrease phagocytosis in vitro of opsonized D+ or D- RBCs. This effect was shown not due to unbound residual chemical, toxicity, or formation of immunoglobulin G oligomers. S-alkylation was required when using low concentrations of reducing compound. CONCLUSION: Our results demonstrate that irreversible reduction of interchain disulfide bonds within the immunoglobulin anti-D results in a significantly increased efficacy to inhibit FcgammaR-mediated phagocytosis regardless of opsonized target cell. With the use of this strategy, more effective and less expensive immunoglobulin treatment for immune cytopenias such as immune thrombocytopenic purpura or autoimmune hemolytic anemia may be developed.