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Mol Cell Biol, May 1998, p. 2486-2491, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Activation of the Small GTPase Ral in Platelets

Rob M. F. Wolthuis,1 Barbara Franke,1,2 Miranda van Triest,1,2 Bettina Bauer,3 Robbert H. Cool,3 Jacques H. Camonis,4 Jan-Willem N. Akkerman,2 and Johannes L. Bos1,*

Laboratory for Physiological Chemistry1 and Department of Hematology,2 Utrecht University, 3584 CG Utrecht, The Netherlands; Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, 44139 Dortmund, Germany3; and U248 INSERM, Section de Recherche, Institut Curie, 75248 Paris Cedex 05, France4

Received 7 November 1997/Returned for modification 18 December 1997/Accepted 1 February 1998

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including alpha -thrombin. In contrast, the platelet antagonist prostaglandin I2 inhibited alpha -thrombin-induced Ral activation. Activation of Ral by alpha -thrombin could be inhibited by depletion of intracellular Ca2+, whereas the induction of intracellular Ca2+ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca2+ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.

* Corresponding author. Mailing address: Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Phone: 31-30-2538989. Fax: 31-30-2539035. E-mail: J.L.Bos{at}med.ruu.nl.

Mol Cell Biol, May 1998, p. 2486-2491, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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