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.
Koo et al. BMC Cell Biology 2007 8:29 doi:10.1186/1471-2121-8-29