Abstract-Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the ␣ heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both ␣ and  subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC. O ne major risk factor for the development of cardiovascular diseases, such as coronary heart disease, stroke, and myocardial infarction, is an imbalance of the production and elimination of reactive oxygen species, also termed as oxidative stress. [1][2][3] As a consequence, the nitric oxide (NO)/ cGMP signaling cascade is impaired, eg, through the excessive production of superoxide, which reacts with NO in a diffusion-limited reaction, yielding peroxynitrite. 4 The biological impact of NO scavenging is further aggravated by the progressive inhibition and downregulation of the NO receptor soluble guanylate cyclase (sGC). [5][6][7][8][9] Circumstantial evidence has implicated proteasomal pathways in this downregulation of sGC. 10 -12 Conversely, a novel class of sGC activators, represented by BAY 58-2667, are potentiated under oxidative stress conditions and represent thus an entirely new diseasespecific vasodilator class. 13 sGC is a heterodimer consisting of an ␣ and a Fe 2ϩ /hemecontaining  subunit that complexes NO with high affinity and specificity. 14 Binding of NO to the Fe 2ϩ /heme results in allosteric activation of the enzyme and enhanced conversion of GTP into the vasorelaxant and antiproliferative second messenger cGMP. 14,15 In vitro experiments have demonstrated that oxidation of sGC heme to its ferric (Fe 3ϩ ) form by the sGC inhibitor 1H-[1,2,4]-oxadiazolo [3,4-a]quinoxalin-1-one (ODQ) attenuates NO-mediated cGMP production, suggesting that the ferro (Fe 2ϩ ) form of sGC is crucial for activation by NO. 16 -18 In addition, studies with primary endothelial and smooth muscle cells have revealed that, within 24 hours, ODQ causes a dramatic decrease in sGC protein levels. 13 Similar results were obtained with other oxidizing compounds such as methylene blue or the peroxynitrite donor 1,3-morpholino-sydnonimine hydrochloride (SIN-1), indicating that oxidative stress triggers downregulation of sGC protein levels. 13 At present, the molecular mechanisms under...
Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is the major cytosolic receptor for NO, catalyzing the conversion of GTP to cGMP. In a search for proteins specifically interacting with human sGC, we have identified the multidomain protein AGAP1, the prototype of an ArfGAP protein with a GTPase-like domain, Ankyrin repeats, and a pleckstrin homology domain. AGAP1 binds through its carboxyl terminal portion to both the ␣ 1 and  1 subunits of sGC. We demonstrate that AGAP1 mRNA and protein are co-expressed with sGC in human, murine, and rat cells and tissues and that the two proteins interact in vitro and in vivo. We also show that AGAP1 is prone to tyrosine phosphorylation by Src-like kinases and that tyrosine phosphorylation potently increases the interaction between AGAP1 and sGC, indicating that complex formation is modulated by reversible phosphorylation. Our findings may hint to a potential role of AGAP1 in integrating signals from Arf, NO/cGMP, and tyrosine kinase signaling pathways.
Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H 2 O 2 induce specific tyrosine phosphorylation of the  1 but not of the ␣ 1 subunit of sGC. Tyrosine phosphorylation of sGC 1 is also inducible by pervanadate and H 2 O 2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of  1 , where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGC 1 , thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.
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