Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding.
We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D009363:Neoplasm Proteins / Q000378:metabolism*; D015689:Oncogene Protein p21(ras); D009857:Oncogenes; D010449:Peptide Mapping; D010957:Plasmids; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin
D009363:Neoplasm Proteins; D013438:Sulfhydryl Compounds; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014357:Trypsin; D015689:Oncogene Protein p21(ras); D005033:Ethylmaleimide