RAF Kinases and Endocytosis Figure 8. Down regulation of A-RAF by either AR149 or A-RAF siRNA interferes with activation of ARF6. A. Interaction of AR149 and ARF6. COS7 cells were co-transfected with GFP-AR149 and either HA-tagged ARF6wt, GTP-locked [ARF6(Q67L)] or GDP-locked [ARF6(T27N)]. After immunoprecipitation with a-GFP antibodies, co-precipitated ARF proteins were detected with a-HA antibodies. In the second experiment, precipitation and detection antibodies were exchanged. Expression levels in whole cell lysates (WCL) is shown in two bottom panels. Empty vectors were used for control (bottom panel). B. GGA3 interacts with ARF6NGTP. COS7 cells were transfected with wild type, GTP-locked (Q67L) or GDP-locked (T27N) HA-ARF6. Proteins pulled-down by incubation with GST-GGA3-Sepharose were detected with a-HA antibodies. C.,E. AR149 suppresses EGF- stimulated ARF6 activation. COS7 cells, transfected with either ARF6 alone or ARF6+GFP-AR149, were treated with EGF for 10 min and subjected to GGA3 pull-down. Bound protein was analysed by immunoblotting. Note decrease in the amount of ARFNGTP by AR149 coexpression. AR149 remains in the pulled-down ARF6 complex confirming the immunoprecipitation data. D. AR149 inhibits ARF6 activation by EFA6. COS7 cells were transfected with HA-ARF6 and either AR149, EFA6, or both. ARF6NGTP was pulled-down by GST-GGA3. AR149 decreases EFA6-stimulated ARF6 activation. F. ARF6 activation by EGF or EFA6 requires A-RAF. ARF6 activation was assessed by GGA3 pull-down. The amount of A-RAF and ARF6 protein were determed by Western blotting (WB). Treatment conditions are as indicated. doi:10.1371/journal.pone.0004647.g008 Figure 9. Model of A-RAF and AR149/DA-RAF function in regulation of endocytosis. Activation of receptor tyrosine kinase (here EGF receptor) leads to RAS-mediated activation of RAF kinases. RAF isoforms sort into different membrane microdomains, such as A-RAF into PtdIns(4,5)P2 rich domains. Activated ERK has opposing effects on A-RAF and C-RAF. Whereas A-RAF is activated, C-RAF becomes inactivated by feedback phosphorylation. A-RAF bound to PtdIns(4,5)P2 rich membranes continues to signal on endosomes leading to ARF6 activation. AR149/DA-RAF locates to recycling endosomes and blocks ERK activation in this compartment. See the main text for details. EE - early endosome, RC – recycling compartment, PM – plasma membrane. Red arrows indicate positive regulation of the process. Pale brown color indicates PtdIns(4,5)P2 rich membrane microdomains. doi:10.1371/journal.pone.0004647.g009 References 1. Rapp UR, Goldsborough MD, Mark GE, Bonner TI, Groffen J, et al. (1983) Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus. Proc Natl Acad Sci U S A 80: 4218–4222. 2. Jansen HW, Lurz R, Bister K, Bonner TI, Mark GE, et al. (1984) Homologous cell-derived oncogenes in avian carcinoma virus MH2 and murine sarcoma virus 3611. Nature 307: 281–284. PLoS ONE | www.plosone.org 17 February 2009 | Volume 4 | Issue 2 | e4647