rasH mutants deficient in GTP binding.

Abstract

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10-to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.The human ras family consists of three known genes (rasH, rasK, and rasN) which have been identified as active transforming genes in a variety of human neoplasms (5). The cellular ras genes encode closely related proteins designated p2is. These plasma-membrane-associated proteins bind guanine nucleotides with high affinity (9,26,32) and display a low GTPase activity (11,19,22,34). Amino acid sequence comparisons have identified homologies between the ras proteins and other proteins which exhibit a high specific affinity for guanine nucleotides, including bacterial elongation and initiation factors, tubulin, and members of the G protein family (16,18). Of these regions of shared sequence homology, the region represented by ras amino acids 110 through 120 is the most striking. We therefore used sitedirected mutagenesis to evaluate the possible role of these ras sequences in guanine nucleotide binding.A comparison of amino acid sequences representing a variety of guanine-nucleotide-binding proteins is shown in Table 1. A consensus sequence of Asn-Lys-X-Asp (human ras residues 116 through 119) was found in all GTP-binding proteins examined. To directly evaluate the involvement of these amino acids in the binding of GTP, oligonucleotidedirected mutagenesis (37, 38) was used to introduce single amino acid substitutions into a cDNA clone of a human rasH gene whose transforming potential was activated by substitution of leucine for glutamine at position 61 (rasH ; 8). Seventeen-base synthetic oligonucleotides, synthesized by the modified triester method, were used to introduce single nucleotide substitutions into a M13mp8 clone containing the rasH cDNA sequence. The following three mutations were isolated: rasH(61-Leu, 116-His), in which codon 116 was changed from AAC (Asn) to CAC (His); rasH(61-Leu, 117-Glu), in which codon 117 was changed from AAG (Lys) to GAG (Glu); and rasH(61-Leu, 119-His), in which codon 119 was changed from GAC (Asp) to CAC (His). The nucleotide sequences of the mutant clones were verified by dideoxy sequencing (31).To evaluate the guanine-nucleotide-binding properties of the mutated rasH proteins, each rasH mutation was constructed into a bacterial expression vector pXVR (8a), which directs synthesis of authentic mammalian p21, and then introduced into Escherichia coli PR13-Q. Upon induction with isopropylthio-3-D-galactoside, approximately 30% of the total bacterial protein was represented by p21. Bacterium-expressed p2is were purified as previously described

An unusual H-Ras mutant isolated from a human multiple myeloma line leads to transformation and factor-independent cell growth

“…Mutations at H-Ras codon 117 have been previously described in chemically induced liver tumors in mice (Anna et al, 1994;Stanley et al, 1994). The observed substitution disrupts a consensus sequence of Asn-Lys-X-Asp (Ras residues 116-119) commonly found in GTP-binding proteins (Der et al, 1986). This region contains the segment of the guanine nucleotide binding pocket including amino acid 117 that interacts with the guanine ring (Figure 7).…”

Differential Effects of a Rab6 Mutant on Secretory Versus Amyloidogenic Processing of Alzheimer's β-Amyloid Precursor Protein

“…In the present study, we used the same approach to examine the possible role of a Golgi-localized Rab protein, Rab6, in the secretory and amyloidogenic processing of ␤APP. By analogy to other members of the Ras superfamily (46,47) WT or Myc-Rab6 N126I ranged from 3-to 5-fold over the endogenous Rab6, as determined by immunoblot analysis with an affinity-purified antibody against Rab6 (not shown). Expression levels of ␤APP 751 were approximately 40-fold over the endogenous ␤APP in 293 cells, so that measurements of products derived from ␤APP (i.e.…”

Recombinant Expression of Caveolin-1 in Oncogenically Transformed Cells Abrogates Anchorage-independent Growth

“…Although the mechanism by which v-Abl activates Ras remains unknown, it has been suggested that v-Abl can interact directly with Shc thereby initiating the formation of a complex with Grb2-mSos and ensuing Ras-Raf activation (49). A mutationally activated form of Ras, most commonly G12V, is found in up to 30% of all human tumors (53)(54)(55)(56)(57)(58)(59)(60)(61).…”