PAS-DEPENDENT MATURATION OF XENOPUS OOCYTES IS BLOCKED BY MODIFIED PEPTIDES OF GTPASE-ACTIVATING PROTEIN (GAP)

Guanosine triphosphatase activating protein (GAP) is an important modu lator of p21(ras) (Ras)-dependent signal transduction in mammalian cel ls and in insulin-induced maturation of Xenopus oocytes. A synthetic o ctapeptide from the catalytic domain of GAP, residues 899-906 (F(899)V FLRLIC(906)), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 mu M). The peptide was as sayed for its ability to block insulin- (Ras-dependent) and progestero ne- (Ras-independent) induced maturation of stage VI Xenopus laevis oo cytes, 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 F-899, GAP(900-906)-NH2, fai led to inhibit GAP-stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine re sulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L-amino acid residues wit h the corresponding D-amino acids produced a peptide with a two-fold i ncreased half-life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modific ations did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP-stimulated Ras GTPase activ ity in vitro were also able to block Pas-dependent GVBD in oocytes, an d (b) specific substitutions in these peptides can result in improved stability in oocytes. (C) Munksgaard 1995.