Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant

Abstract

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 wildtype 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.

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

“…Thus, they do not sequester effectors and are less prone to artifacts that may affect the interpretation of results. A rapid cycling Arf6 mutant, T157A, has been previously characterized (19). For this study, we generated a corresponding Arf5 mutant, Arf5(T161A), which also exhibits enhanced binding to the effector protein GGA3, similar to its Arf6 counterpart (Fig.…”

“…For this study, we generated a corresponding Arf5 mutant, Arf5(T161A), which also exhibits enhanced binding to the effector protein GGA3, similar to its Arf6 counterpart (Fig. 3E) (19).…”

ARF6 and EFA6A Regulate the Development and Maintenance of Dendritic Spines

“…The ARF6 mutants used included dominant-negative ARF6 T27N, which is defective in GTP binding, and fast cycling T157A, which is spontaneously more active than the wild-type protein (Santy, 2002). The latter mutant binds and releases GTP more quickly and induces phenotypes characteristic of ARF6 activation without disrupting the normal functions of the protein (Donaldson, 2003).…”

The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

“…7C, lane 2). The same was true for the activated form of Arf1 (Arf1 T161A), which was generated based on a previously reported fast-cycling Arf6 mutant (28). However, interestingly, when the activated Arf1 and Arf6 mutants were co-expressed in NIH3T3 cells, colony formation in soft agar was enhanced (Fig.…”

“…The QuikChange TM Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutants of Cat-1 that are defective in binding Cool-1 (D294K/E295R) (24), paxillin (K663E/K758E) (25), or are defective as an Arf-GAP (R39A) (26). Likewise, dominant active forms of Arf1 (Q71L, T161A) (27,28) and Arf6 (D125N) (29), as well as dominant negative Arf6 (T27N) (30), were also generated.…”