Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

Abstract

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP‐binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)‐containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6‐23 and 152‐165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165‐184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5‐23 and 152‐165 of ras p21 protein are probably directly involved in the GTP‐binding activity; (ii) GTP‐binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto‐oncogene product; and (iii) the variable region at the C‐terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation.

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product

“…This portion of the K-ras protein sequence is the same in human and mouse DNA and the Kirsten murine sarcoma virus. The first two-thirds of the homologous portion of the ras protein are proposed to be one of the conserved domains necessary for transforming activity and possibly for GTP binding (22). In fact, this portion of the PGP B protein sequence is also homologous to the corresponding H-ras, N-ras, and yeast ras protein sequences, and the difference in the extent of homology mainly reflects the sequence difference in the last one-third of the variable portion of the ras genes.…”

ras p21 deletion mutants and monoclonal antibodies as tools for localization of regions relevant to p21 function.

“…Moreover, deletions of very small stretches at the amino terminus of the molecule, from position 5 to 23, were enough to eliminate GTP-binding activity, whereas a p21 fusion protein in which the first 4 amino acids were substituted by 8 amino acids encoded by the expression vector exhibited GTP-binding activity (25). These findings help to further localize GTP-binding regions of Recognition levels comparable to those of a pAb against the complete p21 protein, which recognized each deletion mutant, are indicated as +.…”

“…The pRC23 plasmid, which carried the PL promoter of X phage and a consensus ribosomebinding site, has been described (23). Construction of expression vectors for p14, p17, p18, p19, and p20 proteins has been described (24,25). The construction of the deletion mutants p8, plO, and p12 was performed as described for p14 and p17 mutants, after digestion with Ava II, Fok I, or Hph I, respectively.…”

H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.

“…1A. Each of the newly generated mutants was placed under the transcriptional control of the Abelson murine leukemia virus long terminal repeat (15), and its transforming activities were analyzed in the NIH 3T3 focusforming assay. The deletion mutant p21 Arg-12 (A58-63), with an oncogenic substitution at position 12, and the deletion mutant p21 Arg-12 Thr-59 (A64-68), with oncogenic substitutions at both positions 12 and 59, showed at least a 100-fold reduction in their respective transforming activities when compared with results for the v-H-ras oncogene p2l Arg-12 Thr-59 ( LG ti 1.1 Hp21-G2T59,A64-68 57 63 69 73…”