Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate,l-arginine. However, the highK_mof arginase (>1 mmol/L) compared with NOS (2 to 20 μmol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification throughS-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS forl-arginine. We demonstrate that arginase1 activity is altered byS-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation.S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces itsK_mvalue 6-fold. Finally, arginase1 nitrosylation is increased (and thus itsK_mdecreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest thatS-nitrosylated arginase1 can compete with NOS forl-arginine and contribute to endothelial dysfunction in the aging cardiovascular system.

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-inducd apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.

The dual-function protein apurinic/apyrmidinic endonuclease/redox factor-1 (APE1/ref-1) is essential for DNA repair and also governs the reductive activation of many redox-sensitive transcription factors. We examined the role of APE1/ref-1 in regulation of endothelium-dependent tone and systemic blood pressure. APE1/ref-1 ^+/− mice have impaired endothelium-dependent vasorelaxation, reduced vascular NO levels, and are hypertensive. APE1/ref-1 upregulates H-ras expression and leads to H-ras-mediated, phosphoinositide-3 kinase/Akt kinase-dependent calcium sensitization of endothelial NO synthase (eNOS), stimulating NO production. The reducing property of APE1/ref-1 is essential for upregulation of H-ras and for the calcium sensitization of eNOS. These findings uncover a novel physiological role for APE1/ref-1 in regulating vascular tone by governance of eNOS activity and bioavailable NO.

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH₂-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.