Carboxyl methylation of Ras regulates membrane targeting and effector engagement.
Post-translational modification of Ras proteins includes prenylcysteine-directed carboxyl methylation. Because Ras participates in Erk activation by epidermal growth factor (EGF), we tested whether Ras methylation regulates Erk activation. EGF stimulation of Erk was inhibited by AFC (N-acetyl-S-farnesyl-L-cysteine), an inhibitor of methylation, but not AGC (N-acetyl-S-geranyl-L-cysteine), an inactive analog of AFC. AFC inhibited Ras methylation as well as the activation of pathway enzymes between Ras and Erk but did not inhibit EGF receptor phosphorylation, confirming action at the level of Ras. Transient transfection of human prenylcysteine-directed carboxyl methyltransferase increased EGF-stimulated Erk activation. AFC but not AGC inhibited movement of transiently transfected green fluorescent protein-Ras from the cytosol to the plasma membrane of COS-1 cells and depleted green fluorescent protein-Ras from the plasma membrane in stably transfected Madin-Darby canine kidney cells, suggesting that methylation regulates Erk by ensuring proper membrane localization of Ras. However, when COS-1 cells were transfected with Ras complexed to CD8, plasma membrane localization of Ras was unaffected by AFC, yet EGF-stimulated Erk activation was inhibited by AFC. Thus, Ras methylation appears to regulate Erk activation both through the localization of Ras as well as the propagation of Ras-dependent signals.
Chiu
Vi K
VK
Department of Medicine, New York University School of Medicine, New York, New York 10016, USA.
Silletti
Joseph
J
Dinsell
Victoria
V
Wiener
Heidi
H
Loukeris
Kristina
K
Ou
Guoming
G
Philips
Mark R
MR
Pillinger
Michael H
MH
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D016827:CD8 Antigens / Q000096:biosynthesis; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008745:Methylation; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
D016827:CD8 Antigens; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine