PROPERTIES OF HUMAN RAS ONCOGENE ENCODED TRANSFORMING PROTEIN

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Project 1: Extension of previous studies on structure and function of ras oncogene-encoded p21 proteins required quantities of purified protein extremely difficult to obtain from eukaryotic cells. To circumvent this problem, column chromatographic procedures were developed to purify normal and transforming p21 proteins from bacterial expression systems. From 1.5 L culture, about 2 mg of apparently homogeneous protein could be obtained. The purified proteins were biochemically active for guanine nucleotide-binding function, autophosphorylation (where applicable) and GTPase activities. Knowledge gained by the studies on GTP-binding function of eukaryotic protein has been extended to purified p21 proteins. p21 proteins with threonine at position 59 show significant GTP binding as compared to the variants with alanine at position 59 without any detectable activity under nonreducing condtions. However, under reducing conditions, p21 proteins with alanine 59 also show enhanced nucleotide binding, although only one-fourth of their threonine counterpart. These results suggest that residue 59 affects the binding of nucleotide to protein. Project 2: Using synthetic antipeptide antibodies corresponding to residues 161-176 in H-ras p21 protein, a structurally and functionally important region in the p21 molecule has been identified which affects the binding of guanine nucleotide to p21 protein. Results with purified p21 proteins and carboxy terminal antibody strongly suggest that the amino and carboxy terminal regions in the p21 molecule are proximal to each other in the native conformation of p21 protein. Project 3: Studies on the characterization of Hs242, a lung carcinoma-derived cell line, have been extended. This cell line, although exhibiting normal phenotype, was found to harbor activated H-ras gene mutated at position 61. Further studies show that H-ras-specific RNA is expressed in the Hs242 cell line. However, analysis of proteins either by immunoprecipitation or by Western blots failed to detect any mutated p21.