A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic Elena Nekhoroshkova, Stefan Albert, Matthias Becker, Ulf R. Rapp* Institut fu¨ r Medizinische Strahlenkunde und Zellforschung (MSZ), University of W u¨rzburg, Wu¨ rzburg, Germany Abstract Background: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. Methodology/Principal Findings: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. Conclusions/Significance: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking. Citation: Nekhoroshkova E, Albert S, Becker M, Rapp UR (2009) A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic. PLoS ONE 4(2): e4647. doi:10.1371/journal.pone.0004647 Editor: Howard Riezman, University of Geneva, Switzerland Received July 15, 2008; Accepted January 13, 2009; Published February 27, 2009 Copyright: 2009 Nekhoroshkova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: EN was supported by DFG grant RA-642/11-2, Deutsch-Franz o¨siches Graduierten kolleg GRK1141/1, SFB 581 Project B5 and SFB 487 Project C3. MB was supported by SFB TR 17 of the DFG. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rappur@mail.uni-wuerzburg.de Introduction RAF protein kinases were originally identified as viral oncogenes [1,2] found in murine and avian retroviruses. RAF genes encode protein serine/threonine kinases [3,4] that mediate transduction of extracellular mitogenic signals from activated Ras GTPases at the plasma membrane to a MAP kinase module (RAF- MEK-ERK), the mitogenic cascade (reviewed in [5]). As a result, complex physiological responses to growth factor stimulation take place at multiple cellular levels. Insect genomes contain only a single RAF gene whereas vertebrates have refined RAF signaling and employ three isoforms that target ERK signalling to different subcellular compartments [6]. Specialized functions are reflected in differential regulation of RAF kinase activity [7] and varying phenotypes of RAF knockout mice [8–10]. The three RAF isoforms and their splice variants share common structural features comprising three conserved regions, CR1, CR2, CR3. The N-terminal CR1 encompasses the Ras binding domain (RBD) and the cysteine-rich domain (CRD), CR2 contains a conserved 14-3-3 binding motif and the C- terminally located CR3 encodes the kinase domain [11]. Whereas B- and C-RAF were studied extensively, little is known about A-RAF function. A-RAF is marked by a low basal kinase activity, which has been attributed to substitutions in its N-region, where tyrosine 296 plays a central role [12]. In contrast to animals with a genomic deletion of B- or C-RAF, A-RAF 2/2 mice are viable, but die perinatally, depending on the genetic background [8]. Budding yeast Saccharomyces cerevisiae is an established eukaryotic model organism that has played a key role in the elucidation of the MAP kinase signaling pathway. Despite the presence of two redundant RAS genes and at least 6 MAPK cascades [13], no RAF kinases are present in S. cerevisiae. Nevertheless, yeast was important for defining RAF function as an activator of a prototypical MAP kinase cascade in experiments that involved ectopic expression of C-RAF [14]. RAF isoforms are known to function as homo and heterodimers in mammalian cells [6], complicating assignment of individual function. Therefore yeast is an attractive system for investigation of a single RAF isoform. Endocytosis is a process essential for many aspects of cellular life, including receptor internalization and recycling. ARF6 GTPase was shown to regulate endocytosis at several levels [15]. The activation state of ARF6 is determined by the bound nucleotide, GTP or GDP, which also affects intracellular localization. Nucleotide loading is regulated by specialized guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze hydrolysis of bound GTP [16]. Endocytosis and signal transduction are known to be functionally linked and regulating each other (Von Zastrow and Sorkin, 2007, Polo and Di Fiore, 2006). ARF6 as a central regulator of endocytic trafficking was shown to activate ERK [17,18]. Based on changes in endocytosis upon inhibition of ERK signaling Robertson et al. (2006) suggested a role of ERK signaling in the regulation of clathrin-independent endocytosis. Here we describe the role of A-RAF in membrane trafficking and identify its function at a specific step of endocytosis. This work PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4647