Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase.
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 H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 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 beta1, 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 sGCbeta1, 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.
Meurer
Sabine
S
Institute for Biochemistry II, University of Frankfurt Medical School, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.
Pioch
Sylke
S
Gross
Steffen
S
Müller-Esterl
Werner
W
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D017968:Vanadium Compounds / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D017968:Vanadium Compounds; C472777:bisperoxovanadium; C076715:pervanadate; D009569:Nitric Oxide; D014638:Vanadates; D014443:Tyrosine; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D019061:src-Family Kinases; D006162:Guanylate Cyclase