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J. Biol. Chem., Vol. 277, Issue 43, 40185-40188, October 25, 2002
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From the Department of Cell Biology, Health Sciences Center,
University of Virginia, Charlottesville, Virginia 22908
Studies of GTPase function often employ
expression of dominant negative or constitutively active mutants.
Dominant negative mutants cannot bind GTP and thus cannot be activated.
Constitutively active mutants cannot hydrolyze GTP and therefore
accumulate a large pool of GTP-bound GTPase. These mutations block the
normal cycle of GTP binding, hydrolysis, and release. Therefore,
although the GTPase-deficient mutants are in the active conformation,
they do not fully imitate all the actions of the GTPase. This is
particularly true for the ADP-ribosylation factors (ARFs), GTPases that
regulate vesicular trafficking events. In Ras and Rho GTPases
replacement of phenylalanine 28 with a leucine residue produces a
"fast cycling" mutant that can undergo spontaneous GTP-GDP exchange
and retains the ability to hydrolyze GTP. Unfortunately this
phenylalanine residue is not conserved in the ARF family of
GTPases. Here we report the design and characterization of a novel
activated mutant of ARF6, ARF6 T157A. In vitro studies show
that ARF6 T157A can spontaneously bind and release GTP more quickly
than the wild-type protein suggesting that it is a fast cycling mutant.
This mutant has enhanced activity in vivo and induces
cortical actin rearrangements in HeLa cells and enhanced motility in
Madin-Darby canine kidney cells.
ACCELERATED PUBLICATION
Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant*
*
This work was supported by National Institutes of Health
Grants F32 DK-09924 (to L. C. S.) and RO1-AI-32991 and RO1-GM-66251 (to James E. Casanova).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: Dept. of Cell Biology,
University of Virginia, P. O. Box 800732, Charlottesville, VA 22908. Tel.: 434-243-5759; Fax: 434-982-3912; E-mail:
ls6e@virginia.edu.
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