title: Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites.
abstract: In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Baljuls
authors.forename: Werner
authors.initials: W
authors.lastname: Schmitz
authors.forename: Thomas
authors.initials: T
authors.lastname: Mueller
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Mirko
authors.initials: M
authors.lastname: Hekman
authors.forename: Ulf R
authors.initials: UR
authors.lastname: Rapp
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D010766:Phosphorylation; D048491:Proto-Oncogene Proteins A-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D048491:Proto-Oncogene Proteins A-raf