Nitric oxide-independent vasodilator rescues heme-oxidized soluble guanylate cyclase from proteosomal degradation

Background: 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 down-regulation of its major intracellular receptor, the alpha/beta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of sGC's heme and responsiveness to NO. Results: 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 down-regulation 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 alpha and beta subunits. Conclusion: 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

Marked elevation in plasma osteoprotegerin constitutes an early and consistent feature of cerebral malaria

Adherence of infected erythrocytes to vascular endothelium causes acute endothelial cell (EC) activation during Plasmodium falciparum infection. Consequently, proteins stored in Weibel-Palade (WP) bodies within EC are secreted into the plasma. Osteoprotegerin (OPG) binds to VWF and consequently is stored within WP bodies. Given the critical role of EC activation in the pathogenesis of severe malaria, we investigated plasma OPG levels in children with P. falciparum malaria. At presentation, plasma OPG levels were significantly elevated in children with cerebral malaria (CM) compared to healthy controls (means 16.0 vs 0.8 ng/ml; p<0.01). Importantly, OPG levels were also significantly higher in children with CM who had a fatal outcome, compared to children with CM who survived. Finally, in children with CM, plasma OPG levels correlated with other established prognostic indices (including plasma lactate levels and peripheral parasite density). To further investigate the relationship between severe malaria and OPG, we utilised a murine model of experimental CM in which C57BL/6J mice were infected with P. berghei ANKA. Interestingly, plasma OPG levels were increased 4.6 fold within 24 hours following P. berghei inoculation. This early marked elevation in OPG levels was observed before any objective clinical signs were apparent, and preceded the development of peripheral blood parasitaemia. As the mice became increasingly unwell, plasma OPG levels progressively increased. Collectively, these data suggest that OPG constitutes a novel biomarker with prognostic significance in patients with severe malaria. In addition, further studies are required to determine whether OPG plays a role in modulating malaria pathogenesis

Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets

Cyclic Nucleotide Dependent Dephosphorylation of Regulator of G-protein Signaling 18 in Human Platelets

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.Science Foundation IrelandUCD School of Medicine and Medical Science, University College Dubli

Dephosphorylation of pS218 of RGS18 in HEK293T cells.

<p>A. Characterization of a pS216pS218 RGS18 double-phospho antibody. FLAG-tagged wt-and point mutants of RGS18 were expressed in HEK293T cells and analyzed by Western blotting using rabbit anti-pS216pS218 RGS18 antibody (upper panel) and subsequently with mouse anti-RGS18 as loading control (lower panel). B. Time-course of S216/S218 phosphorylation and binding to 14-3-3. Wt-RGS18 was expressed in HEK293T cells and before lysis cells were treated with 10 µM forskolin for indicated times. GST-14-3-3γ pull-down assays were performed and precipitates were analyzed by Western blotting using mouse anti-RGS18 (upper panel). Lysates were subjected to Phos-tag supplemented SDS-PAGE and Western blotting using rabbit anti-pS218pS216 RGS18 (lower panel) and subsequently with mouse anti-RGS18 (middle panel). C. Evaluation of three independent experiments for pS216/pS218 (B) and pS216 (blots not shown). Blots were quantified by densitometry and relative signals of pS216/pS218 and pS216 RGS18 were normalized to the highest value. To control for loading, these values were divided by the sum of s1, s2 and s3 bands of the corresponding total RGS18 lanes, normalized to the 0 min time point. Shown are means +SEM. D. Quantitation of results shown in B, top panel. Blots of 2 independent experiments were analyzed by densitometry and relative intensities of precipitated 14-3-3 bound RGS18 versus total RGS18 are presented as means +SEM.</p

RGS18 is regulated by 14-3-3 binding

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.Science Foundation IrelandInternational Society for Advancement of Cytometry (ISAC) Scholar progra

Model of the regulation of the RGS18 complex in platelets.

<p>In resting platelets RGS18 is attached to the scaffold protein spinophilin which also binds PP1, and the tyrosine phosphatase SHP-1 (not shown). In addition, RGS18 binds 14-3-3 via phosphorylated S218 of RGS18 (top). Platelet activation by thrombin, thromboxane A2, or ADP leads to the phosphorylation of S49 and increased 14-3-3 binding. Thrombin also induces the detachment of spinophilin together with PP1, which might prevent dephosphorylation of the 14-3-3 binding sites pS49 and pS218. 14-3-3 reduces the function of RGS18 resulting in facilitated Gq signaling which contributes to platelet activation. In contrast, activation of cAMP- and cGMP-dependent protein kinases by prostacyclin (PGI₂) or nitric oxide (NO) leads to the phosphorylation of RGS18 on serine 216 (pS216). S216 phosphorylation might activate PP1 leading to dephosphorylation of both 14-3-3 binding sites, S49 and S218, and detachment of 14-3-3. Removal of 14-3-3 activates RGS18 to turn off Gq signaling thus contributing to platelet inhibition.</p

Dephosphorylation of pS218 of RGS18 in human platelets treated with forskolin.

<p>A. Time-course of S216 and S218 phosphorylation and binding to 14-3-3. Washed platelets were incubated without or with 10 µM forskolin for the indicated times. Platelets were lysed and lysates were subjected to GST-14-3-3γ pull-down. Precipitates were analyzed by Western blotting using mouse anti-RGS18 antibody (upper panel, RGS18). Lysate blots were incubated with either rabbit anti-pS216pS218 RGS18 (second panel), rabbit anti pS216 RGS18 (third panel) or rabbit anti-RGS18 (lower panel, total RGS18). Shown is a representative example of four independent experiments. B. Quantitation of results shown in A, top panel. Blots of 4 independent experiments were analyzed by densitometry and relative intensities of precipitated RGS18 versus total RGS18 are presented as means +SEM. C. Quantitation of results shown in A, pS216pS218 and pS216 RGS18 panels. Band intensities were determined by densitometry and ratios of pS216/pS218 RGS18 or pS216RGS18 to total RGS18 were calculated. Values were normalized to highest values and presented as means +SEM. D. Effect of phosphatase inhibition on S216/S218 phosphorylation. Washed platelets were incubated without or with 10 µM forskolin or 1 µM okadaic acid as indicated. Platelets were lysed and lysates were subjected to SDS-PAGE and Western blotting. Blots were incubated with either rabbit anti-pS216pS218 RGS18 (upper panel) or rabbit anti-RGS18 (lower panel).</p

Dephosphorylation of pS49 of RGS18.

<p>A. Characterization of an anti-pS49 RGS18 antibody. FLAG-tagged wt and point mutants (as indicated) of RGS18 were expressed in HEK293T cells. Cells were lysed, one sample of wt-expressing cells was incubated with lambda phosphatase for 1h, and all samples were subjected to Phos-tag supplemented SDS-PAGE and Western blotting using mouse anti-total RGS18 antibody (left panel) followed by rabbit anti-pS49 RGS18 antibody (right panel). Appearing bands were labelled as baseline (‘b’), shift 1 (‘s1’), shift 2 (‘s2’), and shift 3 (‘s3’) with increasing apparent molecular weight. B. S49-RGS18 phosphorylation in intact human platelets. Washed platelets were incubated without or with 0.1 U/ml thrombin for 30sec, 1 µM U46619 or 10 µM ADP for 1 min, 10 µM sodium nitroprusside for 10min, 10 µM forskolin for 30min, or a combination of forskolin and thrombin. After lysis and SDS-PAGE, blots were incubated with either anti-pS49 RGS18 (upper panel) or rabbit anti-RGS18 (lower panel). C. Time-course of S49 and S216 phosphorylation of RGS18 in HEK293T cells. Wt-RGS18 transfected HEK293T cells were stimulated with 10 µM forskolin for the indicated times. Cells were then lysed and lysates were subjected to SDS-PAGE and Western blotting using anti-pS49 RGS18 (top panel), rabbit anti-pS7 Rap1GAP2 which detects S216 RGS18 (middle panel) and mouse anti-RGS18 (lower panel). Shown is a representative example of experiments performed three times. D. Quantitation of pS49 and pS216 signals shown in C. Band intensities were determined by densitometry and ratios of pS49-RGS18 or pS216-RGS18 to total RGS18 were calculated. Values were normalized to 0 min forskolin stimulation (pS49) or highest signal (pS216) and presented as means +SEM. Statistical significance in relation to the ‘0’ timepoint was determined using paired Students t-test, *p<0.05. E. Role of S216 for dephosphorylation of pS49 and loss of RGS18 binding to 14-3-3. FLAG-tagged wt- and S216A mutant RGS18 were expressed in HEK293T cells. Cells lysates were subjected to GST-14-3-3γ pull-down. Western blots of precipitates were incubated with mouse anti-RGS18 (upper panel) and lysate blots with rabbit anti-pS49 RGS18 (middle panel) or mouse anti-RGS18 as loading control (lower panel).</p

Slp1 regulates platelet secretion

The small guanine-nucleotide–binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O–permeabilized platelets stimulated with calcium or guanosine 5′-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1.Science Foundation IrelandDeutsche ForschungsgemeinschaftExcellence Cluster Cardio-Pulmonary Syste