Irit Marbach - Academia.edu (original) (raw)

Papers by Irit Marbach

Journal of Biological Chemistry, 1990

, 6579-6583) from our laboratory suggest that G. or a, remain associated with the catalytic subun... more , 6579-6583) from our laboratory suggest that G. or a, remain associated with the catalytic subunit of adenylyl cyclase (C) throughout the activation cycle of adenylyl cyclase by hormone receptors. In this study we have purified GppNHpactivated bovine brain adenylyl cyclase over 3000-fold under mild solution conditions. We demonstrate that although the enzyme is permanently activated it retains the j3 subunit when bound to a forskolin-agarose affinity column as long as it is not exposed to high salt concentrations. The stoichiometry of a, to /3 to C is close to unity, suggesting that @r subunits do not dissociate from G, upon its activation. The complex y&(GppNHp). C dissociates partially when migrating on a Superose 12 fast protein liquid chromatography molecular-seiving column. This partial dissociation probably results from the relatively diluted state of the enzyme at a high degree of purity. Prolonged ultracentrifugation of the complex also causes partial dissociation of the & subunits from cr,(GppNHp). C. The apparent contradiction between the results reported here and the observation that fir subunits inhibit cyclase activity when added to platelet membranes

BioTechniques, 2000

The expression of foreign proteins in Saccharomyces cerevisiaeis a powerful tool for basic resear... more The expression of foreign proteins in Saccharomyces cerevisiaeis a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae , only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in combination with an increase in gene dosage. Most vectors are maintained as plasmids, which forces growth of cultures on poor selective media. Expression of the yeast Gcn4 protein is regulated at the translational level and increases strongly under amino acid starvation. Because under these conditions protein synthesis in general ceases, it is conceivable that regulatory elements that control Gcn4 expression could support selective expression of foreign genes. We cloned DNA fragments residing upstream from the GCN4coding sequence (including the 5 ′ UTR) and ligated them to a cDNA that encodes the human serum albumin (HSA) gene. These GCN4regulatory elements induced efficient HSA expression at the translational level under amino acid starvation. The GCN4 / HSA cassette promoted efficient, inducible expression on either a multicopy or integrative plasmid. The integrated cassette induced a high level of HSA in dense cultures grown on rich media. Thus, the GCN4-based expression system (pGES) provides high protein quantities. pGES is the first expression vector to be induced at the translational level.

Many protein kinases require phosphorylation at their activation loop for induction of catalysis.... more Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2D cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2D cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-med...

In vivo screening of compounds for potential pharmacological activity is more advantageous than i... more In vivo screening of compounds for potential pharmacological activity is more advantageous than in vitro screening. In vivo screens eliminate the isolation of compounds that cannot cross biological membranes, are cytotoxic, or are not specific to the target. However, animal-based or even cell-based systems are usually expensive, time-consuming, and laborious. Here we describe the identification of inhibitors of the mitogen-activated protein kinase p38 via a high throughput screen using yeast cells. p38 is hyperactive in inflammatory diseases, and various indications suggest that its inhibition would reverse inflamma-tion. However, there are currently no p38 inhibitors in clinical use. Because the human p38 imposes severe growth retar-dation when expressed in yeast, we screened a library of 40,000 randomly selected small molecules for compounds that

Molecular Pharmacology

In vivo screening of compounds for potential pharmacological activity is more advantageous than i... more In vivo screening of compounds for potential pharmacological activity is more advantageous than in vitro screening. In vivo screens eliminate the isolation of compounds that cannot cross biological membranes, are cytotoxic, or are not specific to the target. However, animal-based or even cell-based systems are usually expensive, time-consuming, and laborious. Here we describe the identification of inhibitors of the mitogen-activated protein kinase p38alpha via a high throughput screen using yeast cells. p38alpha is hyperactive in inflammatory diseases, and various indications suggest that its inhibition would reverse inflammation. However, there are currently no p38alpha inhibitors in clinical use. Because the human p38alpha imposes severe growth retardation when expressed in yeast, we screened a library of 40,000 randomly selected small molecules for compounds that would restore a normal growth rate. We identified two compounds; both share a structural motif of 4-benzylpiperidine, ...

Biochem J, 2009

MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under o... more MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under optimal growth conditions, their activity is kept off, but in response to stimulation it is dramatically evoked. Because of the high degree of evolutionary conservation at the levels of sequence and mode of activation, MAPKs are believed to share similar regulatory mechanisms in all eukaryotes and to be functionally substitutable between them. To assess the reliability of this notion, we systematically analysed the activity, regulation and phenotypic effects of mammalian MAPKs in yeast. Unexpectedly, all mammalian MAPKs tested were spontaneously phosphorylated in yeast. JNKs (c-Jun N-terminal kinases) lost their phosphorylation in pbs2Delta cells, but p38s and ERKs (extracellular-signal-regulated kinases) maintained their spontaneous phosphorylation even in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Kinase-dead variants of ERKs and p38s were phosphorylated in strains lacking a single MEK (MAPK/ERK kinase), but not in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Thus, in yeast, p38 and ERKs are phosphorylated via a combined mechanism of autophosphorylation and MEK-mediated phosphorylation (any MEK). We further addressed the mechanism allowing mammalian MAPKs to exploit yeast MEKs in the absence of any activating signal. We suggest that mammalian MAPKs lost during evolution a C-terminal region that exists in some yeast MAPKs. Indeed, removal of this region from Hog1 and Mpk1 rendered them spontaneously and highly phosphorylated. It implies that MAPKs possess an efficient inherent autoposphorylation capability that is suppressed in yeast MAPKs via a C-terminal domain and in mammalian MAPKs via as yet unknown means.

Molecular and Cellular Biology, 1992

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regu... more The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a 13-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, -3-fold less potently, the Mn2'-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Va-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyrl proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of -150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the 1B-galactosidase-Cdc25 fusion protein.

Biochemistry, Jan 5, 1999

Saccharomyces cerevisiae Cdc25 is the prototype Ras GDP/GTP exchange protein. Its C-terminal cata... more Saccharomyces cerevisiae Cdc25 is the prototype Ras GDP/GTP exchange protein. Its C-terminal catalytic domain was found to be highly conserved in the homologues p140(ras-GRF) and Sos. The regulatory domains in each Ras exchanger mediate the signals arriving from upstream elements such as tyrosine kinases for Sos, or Ca2+ and G proteins for p140.(Ras-GRF) In this study, we show that the N-terminal half (NTH) of S. cerevisiae Cdc25, as well as the C-terminal 37 amino acids, is essential for processing the elevation of cAMP in response to glucose. The mammalian p140(ras-GRF) catalytic domain (CGRF) restores glucose signaling in S. cerevisiae only if tethered between the N-terminal half (NTH) of S. cerevisiae Cdc25 and the C-terminal 37 amino acids. The glucose-induced transient elevation in cAMP is nullified or severely hampered by the deletion of domains within the NTH of Cdc25. These deletions, however, do not modify the intrinsic GDP/GTP exchange activity of mutant proteins as compa...

European Journal of Biochemistry, 1988

Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC-membranes, wh... more Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC-membranes, which lack G,, induces a threefold reduction of isoproterenol affinity to the P-adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on /3-agonist affinity to their P-adrenoceptors. Non-hydrolyzable analogs such as GTP[S] induce a larger decrease in P-adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis-toxin-catalyzed ADP-ribosylation of Gi. These results suggest that Gi affects P-adrenoceptor affinity to its agonist and that this interaction requires the presence of G,. It seems, therefore, that Gi physically interacts with G, to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis-toxin-catalyzed ADPribosylation in S49 cells on P-agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and CTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of P-agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor-ligand interactions.

European Journal of Biochemistry, 1995

We have previously implemented a combined genetichiochemical approach, for analysis of insertiond... more We have previously implemented a combined genetichiochemical approach, for analysis of insertiondeletion mutants, to identify sites of Harvey-Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger. We showed that positions 101-106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction. Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitutions had no effect on coupling to adenylyl cyclase. Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch I1 region (amino acids 69-78). In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cyclase activation. These findings establish the crucial role of the 102-105 region in the catalyzed exchange reaction and suggest that the 69-74 area would be required for the functional interaction with both exchangers and effector molecules.

European Journal of Biochemistry, 1993

We have cloned, by functional complementation of the cdc2.5-2 mutation of Saccharomyces cerevisia... more We have cloned, by functional complementation of the cdc2.5-2 mutation of Saccharomyces cerevisiae, a homolog of CDC2.5 from the pathogenic yeast Candida albicans. The new gene, named CSC2.5, codes for a 1333-amino-acid protein. The full length gene, as well as a truncated form coding for 795 amino acids, suppresses the thermosensitive phenotype of cdc25" mutants. Biochemical analysis has shown that Csc25 activates the Ras/adenylyl cyclase pathway in S. cerevisiae at a rate two to three times faster than Cdc25, under the same conditions. The C-terminal domain of Csc25 is highly similar to the C-terminal domain of Cdc25, to almost the same extent as the Cterminus of the endogenous Cdc25 homolog Sdc25. We show that polyclonal anti-Cdc25 antibodies interact with Csc25 expressed in S. cerevisiae. In addition to the full length protein (= 150 m a ) , we have found a = 50-kDa polypeptide which seems to include the C-terminus of the CSC25 gene product.

European Journal of Biochemistry, 1992

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guan... more The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[p,y-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the py subunits copurify with the cyclase. The stoichiometry of the cyclase to the a subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (a,) to the p subunit is close to unity, demonstrating that the ,cSy subunits do not dissociate from the G, . cyclase complex (G,, guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the py subunits could be separated from the a, . cyclase complex. Previously reported results on bovine brain cyclase also showed that the G, . cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J . Bid. Chem. 265,9999 -10 0041. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that fly subunits are not released from G, upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosinenucleotide-binding regulatory protein cannot be via shifting the a, to by equilibrium as is commonly believed, and an alternate hypothesis is proposed.

Proceedings of the National Academy of Sciences, 1996

The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response p... more The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure-function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.

Proceedings of the National Academy of Sciences, 1995

Although both Rasl and Ras2 activate adenylyl cyclase in yeast, a number of differences can be ob... more Although both Rasl and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RASJ-RAS2 orRAS2-RAS] genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts rasi ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2A, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than RaslA, in which the C-terminal domain of Rasl has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Rasl, Rasl-Ras2, RaslA, Ras2, and Ras2-Rasl strains throughout the growth curve. This was found to result from the higher expression of Rasl and Rasl-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Rasl and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent Cterminal domains are responsible for these differences, through interaction with other regulatory elements.

PLoS ONE, 2012

Physiologia Plantarum, 1979

Physiologia Plantarum, 1976

... Under such conditions it may be essential to utilize existing storage Page 5. 130 IRITH MARBA... more ... Under such conditions it may be essential to utilize existing storage Page 5. 130 IRITH MARBACH AND AM MAYER Physiol. Plant. 38. ... Plant Physiol. 41: 1455-1458. Chibnall, AC Rees, MW &Williams, EF 1943. The total nitrogen content of egg albumin and other proteins. ...

Journal of Biological Chemistry, 1990

BioTechniques, 2000

Molecular Pharmacology

In vivo screening of compounds for potential pharmacological activity is more advantageous than i... more In vivo screening of compounds for potential pharmacological activity is more advantageous than in vitro screening. In vivo screens eliminate the isolation of compounds that cannot cross biological membranes, are cytotoxic, or are not specific to the target. However, animal-based or even cell-based systems are usually expensive, time-consuming, and laborious. Here we describe the identification of inhibitors of the mitogen-activated protein kinase p38alpha via a high throughput screen using yeast cells. p38alpha is hyperactive in inflammatory diseases, and various indications suggest that its inhibition would reverse inflammation. However, there are currently no p38alpha inhibitors in clinical use. Because the human p38alpha imposes severe growth retardation when expressed in yeast, we screened a library of 40,000 randomly selected small molecules for compounds that would restore a normal growth rate. We identified two compounds; both share a structural motif of 4-benzylpiperidine, ...

Biochem J, 2009

Molecular and Cellular Biology, 1992

Biochemistry, Jan 5, 1999

European Journal of Biochemistry, 1988

European Journal of Biochemistry, 1995

European Journal of Biochemistry, 1993

European Journal of Biochemistry, 1992

Proceedings of the National Academy of Sciences, 1996

Proceedings of the National Academy of Sciences, 1995

PLoS ONE, 2012

Physiologia Plantarum, 1979

Physiologia Plantarum, 1976