We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Two-component systems that enable bacteria to adapt efficiently to changes in the environment consist of two proteins-the transmitter, a protein kinase, and the receiver, the response regulator, which in most cases is a transcriptional activator (reviewed in refs. 1 and 2).One of the best-studied systems is the one responsible for control of the expression of genes in response to the availability of a nitrogen source (reviewed in ref. 3). In this case the kinase, nitrogen regulator II (NR1I), responds to nitrogen deprivation by phosphorylating nitrogen regulator I (NRI); NRI-phosphate binds to sites usually situated -"100 base pairs (bp) upstream of the nitrogen-regulated promoters and catalyzes the conversion of a o-54-RNA polymerase promoter closed complex to the open complex. The core of this system is the glnALG (glnA ntrBC) operon with genes coding, respectively, for glutamine synthetase, NR1I(NtrB) and NRI(NtrC).Three strong NRI binding sites are associated with this operon (4, 5). Two of these sites (sites 1 and 2), situated on the same face of the DNA helix with a center-to-center distance of 31 bp, overlap the a70-dependent promoter ginApi and are 100 bp distant from the o54-dependent promoter glnAp2. A third NRI binding site (Lp) overlaps the or70-dependent promoter glnLp. It has been shown that in cells growing with excess nitrogen, the transcription of ginA (the structural gene for glutamine synthetase) initiates at MATERIALS AND METHODS Proteins. NRI and NR112302 were prepared as described (8,9). The concentrations of NRI and NR112302 were estimated from their absorbances at 280 nm, using Al% = 9.1 for NRI and A1% = 4.15 for NR1I (10). In contrast to previous studies (11,12), the concentrations of NRI are expressed in terms of the dimer. Core RNA polymerase and o54 were purified as described (9).Oligonudeotides. All binding sites were synthesized as 26-bp oligonucleotides and were radioactively end-labeled with the Klenow fragment of Escherichia coli DNA polymerase I (New England Biolabs). End-labeled DNA was separated from unincorporated nucleotides by polyacrylamide gel electrophoresis. For the experiments with the adjacent binding sites 1 and 2 of ginAp2, the 173-bp EcoRI/Sph I fragment from pVW4 carrying these sites was purified by polyacrylamide gel electrophoresis.

Mutations in the gene coding for the renal tight junction protein claudin 16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal Ca(2+) and Mg(2+) handling that progressively leads to chronic renal failure, with nephrolithiasis having been reported in heterozygous carriers. Screening a cohort of 11 families with idiopathic hypercalciuria identified a novel homozygous mutation in the claudin 16 gene in two families. In contrast to classical symptoms of familial hypomagnesemia with hypercalciuria and nephrocalcinosis, the patients displayed serious but self-limiting childhood hypercalciuria with preserved glomerular filtration rate. The mutation results in inactivation of a PDZ-domain binding motif, thereby disabling the association of the tight junction scaffolding protein ZO-1 with claudin 16. In contrast to wild-type claudin 16, the mutant no longer localizes to tight junctions in kidney epithelial cells but instead accumulates in lysosomes. Thus, mutations at different intragenic sites in the claudin 16 gene may lead to particular clinical phenotypes with a distinct prognosis. Mutations in claudin 16 that affect interaction with ZO-1 lead to lysosomal mistargeting, providing-for the first time, to our knowledge-insight into the molecular mechanism of a disease-associated mutation in the claudin 16 gene.

Edited by Roger J. ColbranPhosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2 BAT ), which consists of two cystathionine ␤-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disclike homodimer of CNNM2 BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg 2؉ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

No abstract

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogenlimiting conditions. The general nitrogen regultory protein (NRO) is also dependent on Ea'M (7). The activator protein NIFA also binds to upstream sites (8,9) and is functionally and structurally similar to NRI (10,11). In contrast to NRI, NIFA has not been purified in active form (12,13). Integration host factor (IHF) binds just upstream from the nifI and nifU promoters and stimulates NIFA-mediated activation (13-15). IHF is a sequence-specific DNA-bending protein, which is involved in gene expression and other processes in E. coli and some of its bacteriophages and plasmids (16).The glnHPQ operon of E. coli, which encodes the components of the high-affinity glutamine transport system, is among the operons whose expression is induced under nitrogen-limiting conditions (17, 18). A promoter with homology to the v.54 promoters, glnHp2, has been identified (19). In this study, we present evidence for the existence of overlapping binding sites for NR1 upstream from the gInHp2 promoter. We also found that IHF binds between the glnHp2 promoter and the NRI binding sites. This system allowed us to study the role of IHF in the activation of transcription by NR1 by using purified components. MATERIALS AND METHODSProteins, Primers, and Materials. Core RNA polymerase, or54, NRI, and NRI1 were purified as described (1,20,21). IHF was a gift from C. Robertson and H. Nash (National Institutes of Health). Primers FC5 (5'-CCACATCATCACA-CAATCG-3'), FC6 (5'-CAGACTTCATAGCATTTCC-3'), and FC7 (5'-GCATCTTCAGGGTATTGCC-3') hybridizing at -217, +50, and -103 (5' position), respectively, and primer FC1 (5'-GCGAGAGATATTCGTGG-3'), which hybridizes to T7 sequences close to the HindIII site of plasmid pTE103 (22), were synthesized at the Biopolymers Laboratory, Howard Hughes Medical Institute, Massachusetts Institute of Technology. The following materials were used: DNase I, Mae II, alkaline phosphatase, and bovine serum albumin, from Boehringer Mannheim; Klenow and other restriction endonucleases or DNA modifying enzymes, from New England Biolabs; radiolabels and Protosol, from DuPont/NEN; ultrapure solution ribonucleotides and Sephadex G-25, from Pharmacia LKB.Construction of Plasmids. All transcription templates were derived from plasmid pTE103, which contains the multicloning site from pUC8 placed upstream from a bacteriophage T7 transcriptional terminator (22). Plasmid pFC50 was constructed by inserting the 540-base-pair (bp) EcoRV/Sac II fragment from pTN240 (18), into the Sma I site of pTE103. The sticky ends of this fragment and of those mentioned below were made blunt by using T4 DNA polymerase. The 540-bp fragment contains the glnHp2 promoter with the upstream regulatory sequences and 66 nucleotides of the glnH coding region. Plasmid pFC54 was constructed by inserting the 180-bp Mae II/Sac II fragment from pTN240 ...