Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation.
BACKGROUND
Many studies implicate Arf6 activity in Rac-mediated membrane ruffling and cytoskeletal reorganization. Although Arf6 facilitates the trafficking of Rac1 to the plasma membrane and in many cases Arf6 activation leads to the activation of Rac1, the details of how Arf6 influences Rac function remain to be elucidated.


RESULTS
We demonstrate in binding assays and by co-immunoprecipitation that GDP-bound Arf6 binds to Kalirin5, a Rho family guanine nucleotide exchange factor, through interaction with the spectrin repeat region. In cells, expression of wild type Arf6 recruits spectrin repeat 5 and Kalirin to the plasma membrane and leads to enhanced Kalirin5-induced ruffling. By contrast, expression of an Arf6 mutant that cannot become activated, Arf6 T27N, still recruits spectrin repeat 5 and Kalirin to membranes but inhibits Kalirin5-induced ruffling in HeLa cells. Kalirin5-induced Rac1 activation is increased by the expression of wild type Arf6 and decreased by Arf6T27N. Furthermore, expression of a catalytically-inactive mutant of Kalirin5 inhibits cytoskeletal changes observed in cells expressing EFA6, an Arf6 guanine nucleotide exchange factor that leads to activation of Rac.


CONCLUSIONS
We show here with over-expressed proteins that the GDP-bound form of Arf6 can bind to the spectrin repeat regions in Kalirin Rho family GEFs thereby recruiting Kalirin to membranes. Although Kalirin is recruited onto membranes by Arf6-GDP, subsequent Rac activation and membrane ruffling requires Arf6 activation. From these results, we suggest that Arf6 can regulate through its GTPase cycle the activation of Rac.
Koo
Tae Hyeon
TH
Laboratory of Cell Biology, NHLBI, NIH, Bethesda, MD 20892, USA. gutae@kfda.go.kr <gutae@kfda.go.kr>
Eipper
Betty A
BA
Donaldson
Julie G
JG
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D020662:Guanine Nucleotide Exchange Factors; C503866:KALRN protein, human; D017346:Protein Serine-Threonine Kinases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020830:rac1 GTP-Binding Protein