Nitric-oxide Synthase-2 Linkage to Focal Adhesion Kinase in Neutrophils Influences Enzyme Activity and β2 Integrin Function

Abstract

Background: Reactive nitrogen species increase by an unclear mechanism with exposure to hyperbaric oxygen, which inhibits neutrophil ␤ 2 integrin adherence. Results: Nitric-oxide synthase-2 activity increases when held in proximity to short filamentous actin by focal adhesion kinase. Conclusion: Protein associations are transient because of actin S-nitrosylation.Significance: Nitric-oxide synthase regulation in neutrophils depends on cytoskeletal protein associations.

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

“…The on-going process appears to be centered on actin polymerization/turnover and inhibited by cytochalasin D. This is similar to a mechanism we reported in other studies, where S-nitrosylation drives a series of steps leading to excessive actin turnover (24,31). VASP exhibits higher affinity for S-nitrosylated sF-actin filaments (26); VASP also bundles Rac proteins in close proximity to sF-actin, and subsequent Rac activation increases actin polymerization, which leads to enhanced binding of FAK and then association of iNOS (25). Activation of iNOS involves linkage to actin via FAK, and results from this investigation indicate that HSP90 is also required.…”

“…Elevated iNOS activity requires an association with filamentous actin (25). Whereas VASP exhibits high affinity for SNO-actin, FAK binding is diminished as more actin is S-nitrosylated (25). These relationships underscore the need for an ongoing actin polymerization process and likely a denitrosylation mechanism.…”

“…Elevated iNOS activity involves linkage to FAK and FAK linkage to sF-actin. Elevated iNOS activity requires an association with filamentous actin (25). Whereas VASP exhibits high affinity for SNO-actin, FAK binding is diminished as more actin is S-nitrosylated (25).…”

“…VASP bundles Rac1, Rac2, PKA, and PKG in close proximity to sF-actin, and subsequent Rac activation increases free actin incorporation into filamentous actin (26). Focal adhesion kinase links to the sF-actin and secondary association of iNOS enhances ⅐ NO synthesis (25). The disordered actin polymerization process is reversed by 8-br-cGMP, which activates PKG outside of the sF-actin pool and phosphorylates VASP.…”

“…With regard to ␤ 2 integrin regulation, S-nitrosylation of cytoplasmic actin (SNO-actin) was shown to increase formation of Triton-soluble actin filaments, leading to alterations in the cytoskeletal network that inhibit ␤ 2 integrin-dependent adherence-associated with exposure to high pressure oxygen (24). Reactive nitrogen species are generated because nitric-oxide synthase type 2 (NOS-2 or iNOS) activation occurs (25). Agents capable of causing S-nitrosylation are generated when ⅐ NO synthesized by iNOS reacts with reactive oxygen species (ROS) that are synthesized by myeloperoxidase that is concurrently activated by the hyperoxic exposure (24).…”