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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
-thrombin. In contrast, the platelet
antagonist prostaglandin I2 inhibited
-thrombin-induced
Ral activation. Activation of Ral by
-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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