We show that the two-component signal transduction system of Escherichia coli, CpxA-CpxR, controls the expression of genes encoding cell envelope proteins involved in protein folding and degradation. These findings are based on three lines of evidence. First, activation of the Cpx pathway induces 5-to 10-fold the synthesis of DsbA, required for disulfide bond formation, and DegP, a major periplasmic protease. Second, using electrophoretic mobility shift and DNase I protection assays, we have shown that phosphorylated CpxR binds to elements upstream of the transcription start sites of dsbA, degP, and ppiA (rotA), the latter coding for a peptidyl-prolyl cis/trans isomerase. Third, we have demonstrated increased in vivo transcription of all three genes, dsbA, degP, and ppiA, when the Cpx pathway is activated. We have identified a putative CpxR consensus binding site that is found upstream of a number of other E. coli genes. These findings suggest a potentially extensive Cpx regulon including genes transcribed by ¢r 7° and (r r, which encode factors involved in protein folding as well as other cellular functions.

ArcA protein bearing an amino-terminal, oligohistidine extension has been purified, and its DNA binding activity has been characterized with or without prior incubation with carbamoyl phosphate. Electrophoretic mobility shift assays and DNase I protection assays indicate that where the phosphorylated form of the ArcA protein (ArcA-P) is expected to act as a transcriptional repressor (e.g., of lctPRD and gltA-sdhCDAB), the effect is likely to be mediated by sequestration of cis-controlling transcriptional regulatory elements. In contrast, in the case of cydAB, for which ArcA-P is expected to function as a transcriptional activator, two discrete binding sites have been identified upstream of a known promoter, and activation from these sites is likely to be mediated by a mechanism typical of the type I class of prokaryotic transcriptional activators. An additional ArcA-P binding site has also been located downstream of the known promoter, and a distinct role for this site in the regulation of the cydAB operon during anoxic growth transitions is suggested. These results are discussed within the framework of an overall model of signaling by the Arc two-component signal transduction system in response to changes in aerobiosis.A bacterial cell possesses numerous two-component signal transduction systems which facilitate adaptation to environmental changes. Such systems typically comprise a membraneassociated sensor kinase and a response regulator which controls gene expression (20). In Escherichia coli, adaptation to the respiratory conditions of growth is mediated by the Arc system, consisting of the arcB-encoded sensor kinase and the arcA-encoded response regulator (24,26,29). To date, some 30 operons which are regulated directly or indirectly by the Arc system have been identified (33,34).Although the expression of Arc-regulated genes varies in response to environmental O 2 , this compound is not thought to be the direct signal detected by the ArcB kinase. Instead, ArcB probably senses the redox state of the cell through detection of an electron transport component in reduced form (25). The net activity of ArcB as a kinase for ArcA is expected to progressively increase during transition from aerobic to anaerobic growth, and the concentration of the phosphorylated form of the ArcA (ArcA-P) is therefore predicted to reach peak levels in anoxic cells. However, significant levels of ArcA-P are apparent in aerobic cells (26,28,34), and differential patterns of expression of the members of the Arc modulon can therefore be attributed, at least in part, to differences in the intrinsic affinity of ArcA-P for DNA binding sites located in the transcriptional regulatory regions of its target operons.Working in concert with the Arc system is the Fnr protein, which acts as a transcriptional activator of genes of anaerobic function (including components of fermentation and anaerobic respiration pathways) and as a repressor of some genes encoding proteins of aerobic function (18,33,47). Although the mechanism by which the Fnr prote...

PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding domains originally described in the receptor tyrosine kinase substrate, Shc. By serial truncation, we show that a 174-residue region of Shc p52 (33-206) has full PTB activity. We also show that a 173-residue region of insulin receptor substrate-1 (IRS-1; residues 144 -316) has related PTB activity. In vitro both domains bind directly to activated insulin receptors. Binding is abrogated by substitution of Tyr-960 and selectively inhibited by phosphopeptides containing NPXY sequences. Phosphopeptide assays developed to compare PTB domain specificities show that the Shc PTB domain binds with highest affinity to ⌿XN␤ 1 ␤ 2 pY motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth factor receptors (⌿ ‫؍‬ hydrophobic, ␤ ‫؍‬ ␤-turn forming); the IRS-1 PTB domain does not bind with this motif. In contrast, both the Shc and IRS-1 PTB domains bind ⌿⌿⌿XXN␤ 1 ␤ 2 pY sequences derived from insulin and interleukin 4 receptors, although specificities vary in detail. Shc and IRS-1 are phosphorylated by distinct but overlapping sets of receptor-linked tyrosine kinases. These differences may be accounted for by the inherent specificities of their respective PTB domains.Insulin binding to the insulin receptor activates it as a substrate kinase, leading to tyrosine phosphorylation of at least two cytoplasmic proteins, IRS-1 1 and Shc (1, 2). IRS-1 is phosphorylated at many tyrosine positions (3), whereas Shc is phosphorylated predominantly at one site in cells (4). Since SH2 domain proteins bind specifically with phosphotyrosyl sites in proteins (5, 6), IRS-1 is capable of multiple interactions with SH2 proteins, including phosphatidylinositol 3-kinase, the phosphatase SH-PTP2, and Grb2, a linker protein upstream of Ras. In contrast, when Shc is phosphorylated in cells, it interacts primarily with Grb2 (7).The phosphotyrosine binding (PTB) domain (also called PID or SAIN domain) was recently found to provide a mechanism for protein binding with phosphotyrosyl sequences, distinct from SH2 domains (8 -11). Perhaps related to the phosphorylation of Shc by many tyrosine kinases, in addition to the insulin receptor, its PTB domain appears to interact with multiple phosphotyrosyl proteins (8 -12). The specificity of the Shc PTB domain can be analyzed by methods analogous to those used previously for SH2 domains. The Shc PTB domain binds with ␤ turn-forming motifs frequently containing phosphorylated NPXY sequences (13-15), in contrast with SH2 domains that bind extended phosphopeptide sequences carboxyl-terminal to phosphotyrosine (pTyr) (5, 6). Since efficient IRS-1 phosphorylation in cells also depends on the phosphorylation of a ␤ turnforming NPXY motif in insulin receptors (16), IRS-1 might contain a related PTB domain (even though IRS-1 and Shc show no extended sequence homology). In yeast two-hybrid experiments, the amino-terminal Ϸ500 residues of IRS-1 direct an interaction between the insulin receptor and IRS-1 that is functionally related to Shc PTB do...

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [␥-32 P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.