A Gi‐independent mechanism mediating Akt phosphorylation in platelets

Xiang, Binggang; Zhang, G.; Liu, J.; Morris, Andrew J.; Smyth, Susan S.; Gartner, T. Kent; Li, Z.

doi:10.1111/j.1538-7836.2010.03969.x

Cited by 30 publications

(29 citation statements)

References 30 publications

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“…Our findings are supported by a recent study reporting that Ca 2ϩ is implicated in P2Y12-independent PI3K activation in thrombin-stimulated platelets. 35 Our results expand these observations, because we have demonstrated herein that Ca 2ϩ -dependent activation of PI3K occurs in integrin signaling and we have identified PI3K␤ as the Ca 2ϩ -regulated PI3K isoform. Interestingly, when platelets are stimulated through the other main collagen receptor, GPVI, PI3K␤ stimulation lies upstream of PLC␥2, and is actually required for efficient PLC␥2 activation.…”

Section: Discussionsupporting

confidence: 72%

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Consonni

Cipolla

Guidetti

et al. 2012

Blood

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Integrin ␣2␤1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3K␤, but not of PI3K␥ or PI3K␣. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3K␤ (PI3K␤ KD ), but occurred normally in PI3K␥ KD platelets. Integrin ␣2␤1 failed to stimulate PI3K␤ in platelets from phospholipase C␥2 (PLC␥2)-knockout mice, and we found that intracellular Ca 2؉ linked PLC␥2 to PI3K␤ activation. Integrin ␣2␤1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLC␥2 and intracellular Ca 2؉ . Whereas activation of Pyk2 occurred normally in PI3K␤ KD platelets, stimulation of PI3K␤ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3K␤ was required for ␣2␤1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin ␣IIb␤3 were reduced after inhibition of PI3K␤ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3K␤ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3K␤ downstream of integrin ␣2␤1, and document a novel role for Pyk2 and PI3K␤ in integrin ␣2␤1 promoted inside-out activation of integrin ␣IIb␤3 and thrombus formation. (Blood. 2012;119(3):847-856) IntroductionClass I PI3Ks are key signaling enzymes that phosphorylate the inositol ring of membrane phospholipids and generate different 3-phosphoinositides, important intracellular messengers that regulate several cellular processes through the downstream activation of the protein Ser/Thr kinase Akt. 1 Circulating blood platelets express all members of the class I PI3K family, which includes the PI3K␣, PI3K␤, PI3K␦, and PI3K␥ isoforms. PI3K activity is essential for platelet aggregation and thrombus formation, 1,2 and therefore these enzymes are potential novel targets for antithrombotic agents. For this reason, it is essential to recognize the precise contribution of every PI3K isoform in platelet activation induced by different extracellular agonists.Pharmacologic and genetic evidence indicates that PI3K␤ plays a predominant role in the regulation of platelet function. [3][4][5][6][7] Selective inactivation of PI3K␤ completely prevents platelet aggregation induced by the collagen receptor glycoprotein VI (GPVI) and reduces occlusive thrombus formation. 4,5 PI3K␤ is also implicated in the platelet response to agonists that stimulate G-protein coupled receptors (GPCRs) such as ADP or thromboxane A 2 (TxA 2 ). 3,4,8,9 Whereas PI3K␦ has been demonstrated to play a minor role in platelet activation, 10 PI3K␣ was recently proposed to be as important as PI3K␤ in GPVI signaling. 7 Similarly, several reports have documented that, in addition to PI3K␤, PI3K␥ is also implicated in GPCR-mediated platelet activation. 4,9,11,12 These observations are indicative of a still poorly appreciated interplay...

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Section: Discussionsupporting

confidence: 72%

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Consonni

Cipolla

Guidetti

et al. 2012

Blood

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“…These results suggested that a secondary role of PAR4 activation was required that was not induced by ADP alone. Furthermore, a recent study shows that PAR4 is capable of stimulating Akt phosphorylation in P2Y12 knock-out platelets (14). Taken together, these results suggest that the mechanisms of Akt activation induced by thrombin receptors versus P2Y12 are different, but synergistic.…”

mentioning

confidence: 51%

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

D'Angelo

Chavez

et al. 2011

Journal of Biological Chemistry

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Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3K␣/␤ subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2؊/؊ mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.Arrestins are cytoplasmic proteins that were originally characterized by their ability to associate with agonist-activated G protein-coupled receptors (GPCRs), 2 mediating their internalization and desensitization (1). More recent studies suggest that arrestins play additional roles in GPCR signaling, by serving as scaffolds to recruit signaling complexes to the receptor, thereby facilitating activation of G protein-dependent and -independent pathways (2, 3). One such arrestinmediated pathway is the PI3K-dependent activation of the Ser-Thr kinase, Akt (4, 5). In fibroblasts, colorectal, and gastric carcinoma cells, arrestins have been found to play a critical role in localizing PI3K to GPCR complexes through an interaction with Src family kinases (SFKs) (6 -8). Perhaps most relevant for platelet agonists, thrombin-stimulated Akt phosphorylation involved activation of both G i and G q : G i -dependent signaling to Akt required ras activation, while G q -dependent Akt activation required arrestin-2 (9).Previous work from our laboratory and others has demonstrated that Akt-dependent pathways contribute to platelet activation by G protein-coupled receptors (10, 11). Yet, the mechanisms leading to Akt activation in platelets remain incompletely defined. Multiple laboratories have demonstrated that thrombin-dependent Akt phosphorylation in platelets is reduced by about 90% in the presence of inhibitors for the G i -coupled ADP receptor, P2Y12, and is blocked by inhibi...

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“…In contrast, when PAR4SFT and P2Y12 are coexpressed, arrestin recruitment to AYPGKFactivated PAR4SFT is abolished, and Akt phosphorylation is inhibited. Although many studies have reported that PAR4 signaling to Akt is P2Y12 dependent (Kim et al, 2006;Li et al, 2008), a recent study demonstrated that PAR4 can independently activate Akt in P2Y12 knockout mice and in COS-7 cells at high agonist concentration (AYPGKF 500-1000 mM; Xiang et al, 2010). In the current study, coexpression of P2Y12 and PAR4 was required to detect PAR4-dependent Akt phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

The Physical Association of the P2Y12 Receptor with PAR4 Regulates Arrestin-Mediated Akt Activation

Khan

Ibrahim

et al. 2014

Mol Pharmacol

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It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gqcoupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

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A Gi‐independent mechanism mediating Akt phosphorylation in platelets

Cited by 30 publications

References 30 publications

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

The Physical Association of the P2Y12 Receptor with PAR4 Regulates Arrestin-Mediated Akt Activation

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