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J. Biol. Chem., Vol. 277, Issue 32, 28972-28980, August 9, 2002
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From the Ral GTPases may be involved in
calcium/calmodulin-mediated intracellular signaling pathways. RalA and
RalB are activated by calcium, and RalA binds calmodulin in
vitro. It was examined whether RalA can bind calmodulin in
vivo, whether RalB can bind calmodulin, and whether calmodulin is
functionally involved in Ral activation. Yeast two-hybrid analyses
demonstrated both Rals interact directly but differentially with
calmodulin. Coimmunoprecipitation experiments determined that
calmodulin and RalB form complexes in human platelets. In
vitro pull-down experiments in platelets and in vitro
binding assays showed endogenous Ral and calmodulin interact in a
calcium-dependent manner. Truncated Ral constructs
determined in vitro and in vivo that RalA has
an additional calmodulin binding domain to that previously described,
that although RalB binds calmodulin, its C-terminal region is involved
in partially inhibiting this interaction, and that in vitro
RalA and RalB have an N-terminal calcium-independent and a C-terminal
calcium-dependent calmodulin binding domain. Functionally,
in vitro Ral-GTP pull-down experiments determined that
calmodulin is required for the thrombin-induced activation of Ral in
human platelets. We propose that differential binding of calmodulin by
RalA and RalB underlies possible functional differences between the two
proteins and that calmodulin is involved in the regulation of the
activation of Ral-GTPases.
Calmodulin Binds RalA and RalB and Is Required for
the Thrombin-induced Activation of Ral in Human Platelets*
§,§, and¶
Department of Oral Biology and
¶ Department of Biochemistry and Medical Genetics, University of
Manitoba, Winnipeg, Manitoba R3E 0W2, Canada
*
This work was supported by Grant MT-15408 from the Canadian
Institutes of Health Research (to R. P. B.) and by a graduate student
fellowship from the University of Manitoba.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: 744 Bannatyne
Ave., Winnipeg, Manitoba R3E 0W2, Canada. Tel.: 204-789-3703; Fax: 204-789-3913; E-mail: bhullar@ms.umanitoba.ca.
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