Dynamic fatty acylation of p21N-ras.
To study the acylation of p21N-ras with palmitic acid we have used cells which express the human N-ras gene to high levels under control of the steroid-inducible MMTV--LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N-ras within 5 min, which continued linearly for 30-60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N-ras, suggesting that this can occur as a late post-translational event. Acylated p21N-ras with a high SDS--PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine-labelled p21N-ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N-ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse--chase studies (t1/2 approximately 20 min in serum-containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation--deacylation cycle for p21N-ras, which may be involved in its proposed function as a signal transducing protein.
Magee
A I
AI
National Institute for Medical Research, London, UK.
Gutierrez
L
L
McKay
I A
IA
Marshall
C J
CJ
Hall
A
A
D000215:Acylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D011905:Genes, ras / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D016283:Proto-Oncogene Proteins p21(ras)
D010169:Palmitic Acids; D011518:Proto-Oncogene Proteins; D019308:Palmitic Acid; D003907:Dexamethasone; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)