The recognition that cellular regulatory circuitry is composed of antagonistic elements has made possible a new approach to the selection of regulatory genes, called the gene-gene interference method. The method was used to identify and isolate genes possibly related in an antagonistic way to protein kinase A. Two such genes were recovered: ART1 encodes a potential regulator of cytokinesis and KAI1 appears to be involved in the Start control. The principles of the gene-gene interference method are discussed, as well as its possible general use for 'walking' within the cellular regulatory networks of eukaryotes.
Absolute quantification of protein expression and post-translational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS — Multiplex Absolute Regressed Quantification with Internal Standards — a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labeled standard peptides to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms.
In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.Cyclic AMP is a key regulatory molecule in the life cycle of the yeast Saccharomyces cerevisiae, being involved in the control of cell proliferation and sporulation. Mutations in the adenylate cyclase CDC35 (12, 13, 15) gene lead to growth arrest at the Gl phase of the cell cycle (15), and to induction of sporulation even though the medium is not appropriate for sporulation of the wild type (16). The phenotype of these mutants can be reverted either by addition of cyclic AMP to the medium (13) or by compensatory changes in the cyclic AMP pathway (11, 13). Also, two oncogene homologs of S. cerevisiae RAS] and RAS2 (7,8,14), were found to code for GTP-GDP-binding proteins (9, 17) and to be involved in the activation of adenylate cyclase by guanyl nucleotides (1,18,19). Still another gene, CDC25, appears to be related to the cyclic AMP pathway. In fact, the phenotype of cdc25(Ts) mutants is very similar to that of cdc35(Ts) mutants (15,16). Moreover, as recently described, the level of cyclic AMP decreases significantly in cdc25(Ts) cells when they are shifted to the restrictive temperature, and the addition of cyclic AMP to the medium suppresses the growth thermosensitivity of these cells (3). We have thus siudied the adenylate cyclase activity in membranes prepared from cdc25(Ts) cells harvested before and after a shift to the restrictive temperature. Results presented here strongly suggest that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides.Essentially two strains were used throughout this work: a cdc25(Ts) mutant and a transformant derivative carrying a centromeric plasmid (YCp5O-2) containing the whole CDC25 with the required nutrients (6). After a concentration of 1.4 x 107 to 1.7 x 107 cells per ml was reached, half of the culture (about 100 ml) was filtered through a nitrocellulose membrane (0.45-,Lm pore size; Millipore Corp.), whereas the other half was shifted to 34°C for 19 min (i.e., 15 min after temperature equilibration) before being filtered. Cell lysates and membranes were prepared (at temperatures not exceeding 22°C), and the adenylate cyclase activity of membranes was assayed, essentially as described previously (4). F...
DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis.
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