Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation - PubMed (original) (raw)
Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation
Tae Hyeon Koo et al. BMC Cell Biol. 2007.
Abstract
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.
Conclusion: 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.
Figures
Figure 1
Kalirin5 interacts with Arf6-GDP. A. Domain structure of Kalirin isoforms and Trio showing SR, common first and second DH-PH motifs. (Also depicted, Sec14 domain, oval and SH2 domain, hexagon). B. Arf6, but not Arf1 or Arf5, binds to SR region of Kalirin. COS7 cells were transfected with C-terminally HA-tagged Arf isoforms, and cell extracts were incubated with GST-KalSR4-7 or with GST. Arfs were detected by immunoblotting with anti-HA antibody. The GST fusion proteins were visualized by Coomassie staining. C. Arf6-GDP co-immunoprecipitates with Kalirin5. FLAG-tagged Kalirin5 was co-expressed with HA-tagged Arf6, Arf6Q67L, Arf6T27N or with Arf1 in COS7 cells. Lysates were immunoprecipitated with FLAG antibody and then immunobloted with HA antibody to detect Arf proteins. D. Expression of EFA6 decreases the interaction between Arf6 and Kalirin5. FLAG-tagged EFA6 was co-expressed with Arf6 and HA-tagged Kalirin5 in COS7 cells and the cell lysates were immunoprecipitated with HA antibody and then immunobloted as indicated. E. Energy depletion increases the interaction between Arf6 and Kalirin5. COS7 cells co-transfected with HA-tagged Arf6 WT and FLAG-tagged Kalirin5 were incubated with 50 mM 2-deoxyglucose and 0.02 % sodium azide to deplete cellular ATP and GTP levels for 0, 15, 30, or 60 minutes prior to immunoprecipitation. For each panel, the graph summarizes data for 3 independent experiments (mean ± s.d.). Statistical comparison using one-way ANOVA. *p < 0.05 and **P < 0.01.
Figure 2
SR5 from Trio can interact with Arf6. COS7 cells were transfected with HA-tagged Arf6 with or without FLAG-tagged EFA6. Cell extracts were incubated with GST-tagged TrioSR5-6 or TrioSR1. Complexes were immobilized on glutathione-Sepharose, extensively washed, and separated by SDS-PAGE together with aliquots of total cell lysates. Detection of bound protein was performed by immunoblotting with anti-HA antibody. The bound GST fusion proteins were visualized by Coomassie staining. Graph summarizes data for 3 independent experiments (mean ± s.d.); **p < 0.01 versus Arf6 and Trio SR5-6 (one way ANOVA).
Figure 3
Arf6 recruits Kalirin5 to the PM and endosomes and enhances ruffling. A, B, D, E. Expression of Kalirin5 (K5), SR5 or Arf6 alone (A) or co-expressed with wild type Arf6 (B), Arf6T27N (D) or EFA6 (E). HeLa cells were transfected with FLAG-tagged Kalirin5 or SR5 alone, or with wild type Arf6, Arf6T27N or EFA6. After 18 h, cells were washed, fixed, and processed for indirect immunofluorescence to detect the localization of the overexpressed proteins. C. Increased association of Kalirin with membrane fraction in Cos7 cells expressing Arf6. Rapid fractionation of membrane and cytosol was performed on cells expressing FLAG-Kalirin5 alone or co-expressed with Arf6. The percentage of total Kalirin that was recovered in the membrane pellet is shown for three independent experiments (mean ± s.d.). **p < 0.01 versus Arf6 and Kalirin5 co-transfectants (one way ANOVA).
Figure 4
Arf6 increases Kalirin5-induced Rac1 activation. COS7 cells were co-transfected with AU5-tagged Rac1 and with FLAG-tagged SR5-9 or FLAG-tagged Kalirin5 and with Arf6 or Arf6T27N. For each sample, total cell lysates were incubated with immobilized GST-Pak-CRIB and bound AU5-Rac (AU5-Rac-GTP) was detected by immunoblotting. Total cell lysates were also analyzed by immunoblotting. Graph depicts amount of AU5-Rac1-GTP relative to that observed in Kalirin 5 expressing cells. Mean ± s.d. of three independent experiments; **p < 0.01 versus Kalirin5 transfectants (one way ANOVA).
Figure 5
Dominant negative Kalirin5 (ND/AA) inhibits EFA6 changes in actin organization. A. HeLa cells were transfected with FLAG-tagged EFA6 alone or with HA-tagged Kalirin5 (ND/AA). After 24 h, cells were fixed and labeled with antibodies to detect EFA6, Kalirin and with rhodamine phalloidin to detect actin. Asterisks indicate cells expressing EFA6 only. Arrows indicate cells co-expressing EFA6 and Kalirin5(ND/AA). B. Quantification of cells with radial stress fibers in control, untransfected cells and in cells transfected with EFA6 alone and EFA6 and Kalirin5 (ND/AA). Means ± s.d. of three independent experiments.
References
- Donaldson JG. Arfs, phosphoinositides and membrane traffic. Biochem Soc Trans. 2005;33:1276–1278. - PubMed
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