Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras.
H-Ras displays dynamic cycles of GTP binding and palmitate turnover. GTP binding is clearly coupled to activation, but whether the palmitoylated COOH terminus participates in signaling, especially when constrained by membrane tethering, is unknown. As a way to compare COOH termini of membrane-bound, lipid-modified H-Ras, palmitate removal rates were measured for various forms of H-Ras in NIH 3T3 cells. Depalmitoylation occurred slowly (t(1/2) approximately 2.4 h) in cellular (H-RasWT) or dominant negative (H-Ras17N) forms and more rapidly (t(1/2) approximately 1 h) in oncogenic H-Ras61L or H-RasR12,T59. Combining this data with GTP binding measurements, the palmitate half-life of H-Ras in the fully GTP-bound state was estimated to be less than 10 min. Slow palmitate removal from cellular H-Ras was not explained by sequestration in caveolae, as neither cellular nor oncogenic H-Ras showed alignment with caveolin by immunofluorescence. Conversely, although it had faster palmitate removal, oncogenic H-Ras was located in the same fractions as H-RasWT on four types of density gradients, and remained fully membrane-bound. Thus the different rates of deacylation occurred even though oncogenic and cellular H-Ras appeared to be in similar locations. Instead, these results suggest that acylprotein thioesterases access oncogenic H-Ras more easily because the conformation of its COOH terminus against the membrane is altered. This previously undetected difference could help produce distinctive effector interactions and signaling of oncogenic H-Ras.
Baker
Tara L
TL
Department of Zoology/Genetics, Iowa State University, Ames, Iowa 50011, USA.
Zheng
Hui
H
Walker
Joy
J
Coloff
Jonathan L
JL
Buss
Janice E
JE
D016475:3T3 Cells; D000215:Acylation; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005347:Fibroblasts / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D011905:Genes, ras / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D019308:Palmitic Acid / Q000378:metabolism*; D010170:Palmitoyl-CoA Hydrolase / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013010:Sonication; D014162:Transfection; D014316:Tritium
C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D014316:Tritium; D006153:Guanosine Diphosphate; D019308:Palmitic Acid; D006160:Guanosine Triphosphate; D010170:Palmitoyl-CoA Hydrolase; D016283:Proto-Oncogene Proteins p21(ras); D003545:Cysteine