Akt Activation in Platelets Depends on Gi Signaling Pathways

Satya P. Kunapuli,Soochong Kim,Jianguo Jin

doi:10.1074/jbc.m306162200

Satya P. Kunapuli, Soochong Kim + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m306162200

Copy DOI

Abstract

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.

Highlights

Akt ( known as protein kinase B (PKB))1 is a 57-kDa serine/threonine kinase, which is the cellular homologue of the ʈ To whom correspondence should be addressed: Dept. of Physiology, Temple University School of Medicine, 3420 N
An increase in Ser473 phosphorylation was detectable at concentrations above 3 ␮M Stimulation of platelets with a PAR1-activating peptide (SFLLRN), 200 ␮M AYPGKF, or 0.1 unit/ml thrombin; higher concentrations revealed further phosphorylation that peaked at concentrations above 5 ␮M SFLLRN, 500 ␮M AYPGKF, or 0.5 unit/ml thrombin
SFLLRN, AYPGKF, and thrombin-induced Akt phosphorylation were inhibited in the presence of the PI 3-kinase inhibitor, LY294002, suggesting that phosphorylation of Akt is PI 3-kinase dependent. These results indicate that secreted ADP and Gi-coupled P2Y12 receptors are important in SFLLRN, AYPGKF, and thrombin-induced phosphorylation of Akt, and PI 3-kinase is a key element in the Gi-coupled pathway for Akt phosphorylation

Summary

Akt Activation in Platelets Depends on Gi Signaling Pathways*

From the ‡Department of Physiology, the §Department of Pharmacology, and the ¶Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets. In human and mouse platelets, we have found that thrombin- and thrombin receptor-activating peptide-mediated

Akt activation is largely dependent on signaling through the

EXPERIMENTAL PROCEDURES

RESULTS

Gi Pathways Activate Akt in Platelets

DISCUSSION