Objetivo A initial genetic approach has allowed the definition of single amino acid residues of the Ras2 protein that are critical for its function. After random mutagenesis of the chromosomal RAS2 gene in ras1 strains, isogenic yeast cells expressing Ras2 proteins with either increased or reduced function have been isolated and characterised. 4 regions of Ras2 have been identified where single amino acid substitutions were capable of deregulating the function of the protein. Theseregions are centred around positions 19, 40, 70 and 82-84. Since the yeast and the human Ras proteins are highly related over the 160 amino terminal amino acids, the positions that have been identified correspond to residues 12, 33, 63 and 75-77 of the human protein.The wild type and mutated Ras2 proteins have been purified using an Escherichia coli expression system. Moreover, yeast membranes used as a source of either Ras or adenylyl cyclase have allowed the performance of in vitro complementation assays between wild type and mutated forms of both enzymes.The relevant conclusions of the biochemical analyses can be summarised as follows:The Ras2 protein is capable of stimulating adenylyl cyclase in its guanosine triphosphate (GTP) bound, but not in its guanosine diphosphate (GDP) bound form. Furthermore, the guanosine diphosphate aminophosphate bound form is as effective as the GTP bound form. Therefore, the guanosine triphosphatase (GTPase) activity of the protein can be considered as a turnoff mechanism for the conversion of the active to the inactive form of Ras.The replacement of amino acids in the region 12-13 or 59-63 by other residues activates the oncogenic function of the protein in high eukaryotes. This is due to an impaired GTPase activity of the protein, that therefore cannot attain the inactive (GDP bound) state. In yeast cells, the same mutations cause an inability to properly arrest in the G1 phase of the cell cycle.The regeneration of the active from the inactive form takes place by a nucleotide exchange reaction, in which the rate limiting step is the dissociation of GDP. Macromolecular effectors like Sdc25 are able to increase the rate of this reaction in vitro, thus catalysing the formation of the active Ras2.GTP complex. The effect of macromolecular effectors on the nucleotide exchange reaction can be mimicked by a single amino acid substitution at position 152 of the Ras2 protein.The biochemical analysis of Ras mutants, that were initially found as temperature sensitive lethals, has provided important clues to the mechanism of guanosine triphosphate (GTP) activation of Ras.The region centred around position 33 is important for the interaction of Ras with its target, since a single amino acid substitution at this position reduced the ability of Ras to stimulate adenylyl cyclase. The structure of this loop is reorganised upon replacement of guanosine diphosphate (GDP) by GTP.Residues 75-77 are also important for function, since the introduction of other amino acids strongly reduced adenylyl cyclase stimulation by Ras.The replacement of the glycine residue 75 by serine did not modify the affinity of Ras for GDP, but it decreased the affinity for the GTP analogue guanosine diphosphate aminophosphate. Therefore, despite the great thermal mobility of loop L4-L5, the modification of the glycine residue 75 can influence the nucleotide binding site. A possible explanation is that loops L4-L5 are mobile in some directions, but that the 2 loops are relatively rigid in linking nucleotide proximal with distal residues. A further implication of this result is that the binding of macromolecular effectors of Ras to loops L4-L5 could influence the binding of the nucleotide, and vice versa. One important aspect of our future work will be the evaluation of functional properties of mutated Ras proteins harbouring additional single amino acid substitutions in loops L2 and L4-L5.The function of the elongation factor Tu (EF-Tu) from bacteria is modulated by the alternate binding of guanosine triphosphate (GTP) or guanosine diphosphate (GDP) during the elongation cycle. This protein has been the object of intensive biochemical analysis, and the mechanism of action of several antibiotics acting on EF-Tu has been elucidated. A new antibiotic acting specifically on the GTP bound form of EF-Tu has now been identified and molecularly characterised. Moreover, the role of specific amino acids in the guanosine triphosphatase (GTPase) reaction has been investigated. Hopefully, the similarity between the molecular mechanisms regulating the function of Ras and EF-Tu will facilitate the production of antibiotics acting specifically on the oncogenic forms of Ras.It has been previously established that the yeast adenylyl cyclase is functionally inactive in the absence of Ras proteins, and the synthesis of cyclic adenosine monophosphate (AMP) is activated by the presence of the guanosine triphosphate (GTP) bound form of Ras. It has also been found that single amino acid substitutions at position 1651 of the adenylyl cyclase resulted in a detectable activity even in the absence of Ras. This could be explained by a model in which regulatory amino terminal regions of adenylyl cyclase negatively and reversibly regulate a carboxy terminal catalytic domain in the absence of Ras; and amino acid residue 1651 is critical for the interaction between regulatory and catalytic domains. According to the model, the Ras independent activation of catalytic function by single amino acid substitutions at position 1651 could be explained by a reduced interaction between inhibitory and catalytic domains.This model predicted that some amino acid substitutions at position 1651 could lead to a stronger interaction with regulatory domains, thus resulting in hyperinhibition of catalytic function. This prediction has been confirmed by the finding that the introduction of negatively charged residues at position 1651 could reduce the weak activation of the catalytic domain that follows the overexpression of the adenylyl cyclase in a ras- background. In agreement with the model, the removal of amino terminal domains form the enzyme carrying a negatively charged residue at position 1651 resulted in reactivation of the function of the catalytic domain.Advantage can be taken of the enzyme carrying a negatively charged residue at position 1651 to select for randomly induced mutations that could reactivate the function of the attenuated adenylyl cyclase. Point mutations have been identified, leading to single amino acid substitutions at positions 1331, 1345, 1348 and 1374, individually capable of reactivating the mutated enzyme. These residues define a new region of adenylyl cyclase that is involved in the negative control of the catalytic domain in the absence of Ras.A mutated adenylyl cyclase harbouring a single amino acid substitution at position 1374 and a wild type sequence at position 1651 was found to show an impaired response to Ras. Therefore, by searching for amino acid residues that are involved in the negative control ofthe catalytic activity in the absence of active Ras proteins, a residue has also been identified that is important for Ras dependent activation. This suggests that some amino acid residues are critical both for the negative regulation of the catalytic centre in the absence of active Ras and for the Ras dependent regulation.Genetic techniques have been used to identify genes that have a functional relationship with Ras. 2 genes have been isolated, originally called PR3E and KOM1, that were able to suppress a temperature sensitive RAS function, upon expression on a high copy number plasmid. From the nucleotide sequence the 2 genes appeared to be alleles of TPK3 and SCH9, respectively. These genes have been previously identified as suppressors of a temperature sensitive CDC25 function. The disruption of SCH9 KOM1, that appears to encode a putative protein kinase of 824 amino acids, resulted in a slower growth rate. It has been found that the levels and the activity of the RAS1, RAS2 and adenylyl cyclase gene products in cells growing logarithmically were not significantly affected by the disruption of SCH9. Moreover, the temperature sensitivity of some RAS2 mutants was increased by disruption of the SCH9 gene. Therefore, SCH9 might be either a downstream element of the RAS adenylyl cyclase pathway, or an element of a parallel pathway. Exploration of the relationship between SCH9 and other elements of the pathway is continuing using by genetic techniques. Ámbito científico natural sciencesbiological sciencesmicrobiologybacteriologynatural sciencesbiological sciencesgeneticsmutationnatural sciencesbiological sciencesgeneticsnucleotidesnatural scienceschemical sciencesorganic chemistryaminesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Programa(s) FP1-STIMULATION 1C - Plan (EEC) to stimulate European scientific and technical cooperation and interchange, 1985-1988 Tema(s) Data not available Convocatoria de propuestas Data not available Régimen de financiación CSC - Cost-sharing contracts Coordinador EUROPEAN MOLECULAR BIOLOGY LABORATORY Aportación de la UE Sin datos Dirección Meyerhofstrasse 1 HEIDELBERG Alemania Ver en el mapa Coste total Sin datos Participantes (2) Ordenar alfabéticamente Ordenar por aportación de la UE Ampliar todo Contraer todo ECOLE POLYTECHNIQUE Francia Aportación de la UE Sin datos Dirección ROUTE DE SACLAY PALAISEAU Ver en el mapa Coste total Sin datos Università degli Studi di Napoli Federico II Italia Aportación de la UE Sin datos Dirección Via Sergio Pansini 5 80131 Napoli Ver en el mapa Coste total Sin datos
