PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.

A novel role for von Willebrand factor in the pathogenesis of experimental cerebral malaria.

Plasmodium falciparum malaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium berghei ANKA. In keeping with findings in children with P falciparum malaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P berghei infection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P berghei infection in VWF(-/-) C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF(-/-) mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF(-/-) and WT mice. Interestingly, however, the degree of ECM-associated enhanced blood-brain barrier permeability was significantly attenuated in VWF(-/-) mice compared with WT controls. Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance.

Galectin-1 and Galectin-3 Constitute Novel-Binding Partners for Factor VIII.

OBJECTIVE: Recent studies have demonstrated that galectin-1 (Gal-1) and galectin-3 (Gal-3) can bind von Willebrand factor and directly modulate von Willebrand factor-dependent early thrombus formation in vivo. Because the glycans expressed on human factor VIII (FVIII) are similar to those of von Willebrand factor, we investigated whether galectins might also bind and modulate the activity of FVIII. APPROACH AND RESULTS: Immunosorbant assays and surface plasmon resonance analysis confirmed that Gal-1 and Gal-3 bound purified FVIII with high affinity. Exoglycosidase removal of FVIII N-linked glycans significantly reduced binding to both Gal-1 and Gal-3. Moreover, combined removal of both the N- and O-glycans of FVIII further attenuated Gal-3 binding. Notably, specific digestion of FVIII high-mannose glycans at N239 and N2118 significantly impaired FVIII affinity for Gal-1. Importantly Gal-1, but not Gal-3, bound to free FVIII in the plasma milieu, and significantly inhibited FVIII functional activity. Interestingly, commercial recombinant FVIII (rFVIII) concentrates are manufactured in different cell lines and differ in their glycosylation profiles. Although the biological mechanism has not been defined, recent studies in previously untreated patients with severe hemophilia A reported significant differences in inhibitor development associated with different rFVIII products. Interestingly, Gal-1 and Gal-3 both displayed enhanced affinity for BHK-rFVIII compared with CHO-rFVIII. Furthermore, binding of Gal-1 and Gal-3 to BDD-FVIII was markedly reduced compared with full-length rFVIII. CONCLUSIONS: We have identified Gal-1 and Gal-3 as novel-binding partners for human FVIII and demonstrated that Gal-1 binding can influence the procoagulant activity of FVIII.

Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets.

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.

Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets.

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.

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

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 alphabeta 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 alpha and beta 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.

Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells.

Signaling by Eph receptors and cell-surface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact-dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5-induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors.

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.