Ras-dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP).
Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)-dependent signal transduction in mammalian cells and in insulin-induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899-906 (F899VFLRLIC906), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 microM). The peptide was assayed for its ability to block insulin- (Ras-dependent) and progesterone- (Ras-independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD). Microinjection of 50 pmol of the peptide inhibited insulin- but not progesterone-induced GVBD by 50%. A 7-residue peptide lacking F899, GAP(900-906)-NH2, failed to inhibit GAP-stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine resulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L-amino acid residues with the corresponding D-amino acids produced a peptide with a two-fold increased half-life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modifications did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP-stimulated Ras GTPase activity in vitro were also able to block Ras-dependent GVBD in oocytes, and (b) specific substitutions in these peptides can result in improved stability in oocytes.
Losardo
J E
JE
Department of Oncology, Hoffmann-La Roche Inc., Nutley, New Jersey, USA.
Heimer
E
E
Bekesi
E
E
Prinzo
K
K
Scheffler
J E
JE
Neri
A
A
D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D020690:GTPase-Activating Proteins; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011374:Progesterone / Q000494:pharmacology; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014982:Xenopus laevis; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007328:Insulin; D009842:Oligopeptides; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D011374:Progesterone; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)