Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

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Characterization and expression of the human rhoH12 gene product

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2058-2066.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2058-2066

Author(s):

H Avraham ◽

R A Weinberg

Keyword(s):

Amino Acids ◽

Cell Lines ◽

Soft Agar ◽

Cdna Clones ◽

Focus Formation ◽

Reductase Gene ◽

Evolutionarily Conserved ◽

Normal Human ◽

Nih 3T3 ◽

P21 Proteins

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

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Abstract WP263: Mutagenesis Studies Revealed Minimal Impact of Human A120T Variant of Protease-activated Receptor 4 on Receptor function or Pharmacological Response to a Potent and Selective Antagonist

Stroke ◽

10.1161/str.47.suppl_1.wp263 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Mario F Callejo ◽

Jeffrey Colin ◽

Sophie Desmeules ◽

Chi Sum ◽

Tao Wang ◽

...

Keyword(s):

African American ◽

Cell Lines ◽

Site Directed Mutagenesis ◽

Hek293 Cells ◽

Cell Surface Expression ◽

Directed Mutagenesis ◽

Platelet Response ◽

Functional Responses ◽

Stroke Treatment

Introduction: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor and a novel target for ischemic stroke treatment. Recent reports suggested a subtle racial difference in platelet responses to submaximal concentrations of PAR4 agonist peptide (PAR4-AP). One of the PAR4 variants, A120T, is more common in African American than white (63% vs. 19%, Edelstein et al., Blood 2014). It was suggested that this variant contributes to the difference in response to PAR4-AP and impacts the in vitro response to YD-3, a PAR4 antagonist. In this study, site-directed mutagenesis was used to evaluate the effect of the A120T variant on PAR4 function and response to a newly discovered potent and selective PAR4 antagonist, UDM-001651. Unlike YD-3, UDM-001651 inhibited thrombin-induced platelet aggregation and prevented thrombosis in a monkey model and thus served as a relevant PAR4 pharmacology tool. Methods: Human PAR4 cDNAs expressing A120 and T120 variants were stably expressed in HEK293 cells. Cell surface expression of PAR4 was analyzed by FACS. Functional responses of cells were evaluated by monitoring calcium mobilization induced by BMS PAR4-AP, which was optimized based on AYPGKF. The potency of UDM-001651 was derived from an 11-point concentration response curve in the calcium assay using PAR4-AP at the EC80 concentration. Transient transfection studies were also performed to confirm the results. Results: Comparable levels of expression and functional responses were observed between A120 and T120 expressing cells. Results from side-by-side comparison between the two cell lines demonstrated no detectable difference in the mean EC50 values (0.42±0.054 versus 0.46±0.051 uM standard error, n=9) in calcium responses to PAR4-AP stimulation. Similarly, the potency of UDM-001651 in the same assay were similar between these two cell lines. Moreover, preliminary clinical results did not show differences in PAR4-mediated platelet response between African-American and white subjects. Conclusion: In contrast to the previous report, the results reported herein from site directed mutagenesis studies indicate that the A120T variant of PAR4 has no apparent impact on calcium signaling in response to agonist stimulation or response to a PAR4 antagonist.

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Mutagenesis of the borage Δ6 fatty acid desaturase

Biochemical Society Transactions ◽

10.1042/bst0280636 ◽

2000 ◽

Vol 28 (6) ◽

pp. 636-638 ◽

Cited By ~ 15

Author(s):

O. Sayanova ◽

F. Beaudoin ◽

B. Libisch ◽

P. Shewry ◽

J. Napier

Keyword(s):

Fatty Acid ◽

Consensus Sequence ◽

Fatty Acid Desaturase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

The Third ◽

Membrane Bound ◽

Fusion Domain ◽

Δ6 Desaturase

The consensus sequence of the third histidine box of a range of Δ5, Δ6, Δ8 and sphingolipid desaturases differs from that of the membrane-bound non-fusion Δ12 and Δ15 desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b5 fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Δ6 desaturase.

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Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1809 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1809-1813 ◽

Cited By ~ 19

Author(s):

R G Chipperfield ◽

S S Jones ◽

K M Lo ◽

R A Weinberg

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Normal Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Transforming Activity ◽

Ras P21 ◽

Ras Oncogenes ◽

Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

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Characterization and expression of the human rhoH12 gene product.

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2058 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2058-2066 ◽

Cited By ~ 78

Author(s):

H Avraham ◽

R A Weinberg

Keyword(s):

Amino Acids ◽

Cell Lines ◽

Soft Agar ◽

Cdna Clones ◽

Focus Formation ◽

Reductase Gene ◽

Evolutionarily Conserved ◽

Normal Human ◽

Nih 3T3 ◽

P21 Proteins

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

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Sequential Autoprocessing of Marek's Disease Herpesvirus Protease Differs from That of Other Herpesviruses

Journal of Virology ◽

10.1128/jvi.02679-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 6117-6121 ◽

Cited By ~ 4

Author(s):

S. Laurent ◽

C. Blondeau ◽

M. Belghazi ◽

S. Remy ◽

E. Esnault ◽

...

Keyword(s):

Escherichia Coli ◽

Consensus Sequence ◽

Marek's Disease ◽

Site Directed Mutagenesis ◽

Viral Capsid ◽

Directed Mutagenesis ◽

Marek’S Disease ◽

Polar Amino Acid ◽

Kinetics Studies ◽

Capsid Maturation

ABSTRACT Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)P3-XP2-AP1/SP1′ (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: MP3-NP2-AP1/SP1′. Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

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Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

Journal of Bacteriology ◽

10.1128/jb.186.3.850-857.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 850-857 ◽

Cited By ~ 8

Author(s):

Geneviève Renauld-Mongénie ◽

Laurence Lins ◽

Tino Krell ◽

Laure Laffly ◽

Michèle Mignon ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Prediction Method ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Directed Mutagenesis ◽

Binding Domains ◽

Homologous Strain ◽

Protein B

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

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Structural analysis of natriuretic peptide receptor-C by truncation and site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3220585 ◽

1997 ◽

Vol 322 (2) ◽

pp. 585-590 ◽

Cited By ~ 5

Author(s):

Makoto ITAKURA ◽

Hiroyuki SUZUKI ◽

Shigehisa HIROSE

Keyword(s):

Ligand Binding ◽

Natriuretic Peptide ◽

Gel Filtration ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Natriuretic Peptide Receptor ◽

Peptide Receptor ◽

Mammalian Expression ◽

Ligand Binding Activity

Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the mammalian expression vector pcDNA3 and expressed by transient transfection in COS-1 cells. Truncation at position 461, which eliminates the residue Cys469 that is involved in disulphide-linked dimerization, produced a soluble and monomeric form of NPR-C, as determined by gel filtration on Superose 12. Binding assays of the gel-filtration fractions clearly demonstrated that even monomeric NPR-C contains a high-affinity binding site for natriuretic peptides. Site-directed mutagenesis of the invariant residues (Asp407-Arg408 and Asp411-Phe412) in a region highly conserved among various species established that these invariant residues are essential for ligand-binding activity.

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Positive and negative regulation of basal expression of a yeast HSP70 gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2025-2033.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2025-2033

Author(s):

H O Park ◽

E A Craig

Keyword(s):

Heat Shock ◽

Consensus Sequence ◽

Point Mutations ◽

Binding Activity ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Basal Expression ◽

Cell Extracts ◽

Dna Binding Assay ◽

Kinetics Of

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.

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