Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.
Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.
Rey
I
I
Rhône-Poulenc Santé Centre de Recherche de Vitry, Vitry Sur Seine, France.
Soubigou
P
P
Debussche
L
L
David
C
C
Morgat
A
A
Bost
P E
PE
Mayaux
J F
JF
Tocque
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000242:Cyclic AMP / Q000096:biosynthesis; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D011506:Proteins / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras); D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D000906:Antibodies; D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)