Identification of key residues in the A-Raf kinase important for phosphoinositide lipid binding specificity.
Raf kinases are involved in regulating cellular signal transduction pathways in response to a wide variety of external stimuli. Upstream signals generate activated Ras-GTP, important for the relocalization of Raf kinases to the membrane. Upon full activation, Raf kinases phosphorylate and activate downstream kinase in the mitogen-activated protein kinase (MAPK) signaling pathway. The Raf family of kinases has three members, Raf-1, B-Raf, and A-Raf. The ability of Raf-1 and B-Raf to bind phosphatidylserine (PS) and phosphatidic acid (PA) has been show to facilitate Raf membrane associations and regulate Raf kinase activity. We have characterized the lipid binding properties of A-Raf, as well as further characterized those of Raf-1. Both A-Raf and Raf-1 were found to bind to 3-, 4-, and 5-monophosphorylated phosphoinositides [PI(3)P, PI(4)P, and PI(5)P] as well as phosphatidylinositol 3,5-bisphosphate [PI(3,5)P(2)]. In addition, A-Raf also bound specifically to phosphatidylinositol 4,5- and 3,4-bisphosphates [PI(4,5)P(2) and PI(3,4)P(2)] and to PA. A mutational analysis of A-Raf localized the PI(4,5)P(2) binding site to two basic residues (K50 and R52) within the Ras binding domain. Additionally, an A-Raf mutant lacking the first 199 residues [i.e., the entire conserved region 1 (CR1) domain] bound the same phospholipids as full-length Raf-1. This suggests that a second region of A-Raf between amino acids 200 and 606 was responsible for interactions with the monophosphorylated PIs and PI(3,5)P(2). These results raise the possibility that Raf-1 and A-Raf bind to specific phosphoinositides as a mechanism to localize them to particular membrane microdomains rich in these phospholipids. Moreover, the differences in their lipid binding profiles could contribute to their proposed isoform-specific Raf functions.
Johnson
Lindsey M
LM
Department of Biochemistry, University of Saskatchewan, 107 Wiggins Avenue, Saskatoon, Saskatchewan, S7N 5E5 Canada.
James
Kristy M
KM
Chamberlain
M Dean
MD
Anderson
Deborah H
DH
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D048491:Proto-Oncogene Proteins A-raf / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D000596:Amino Acids; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C060974:phosphatidylinositol 3,4,5-triphosphate; D048491:Proto-Oncogene Proteins A-raf; D019908:Proto-Oncogene Proteins c-raf