Mark Donowitz

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins

The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ (transcriptional co‐activator with PDZ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

Na + /H + exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na+/H+ exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase Cβ1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2–PTH1R complex markedly activates phospholipase Cβ and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (Gi/o proteins). NHERF-mediated assembly of PTH1R and phospholipase Cβ is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.

Differential roles of NHERF1, NHERF2, and PDZK1 in regulating CFTR-mediated intestinal anion secretion in mice

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta2-adrenergic receptor (beta2-AR) stimulation. Conversely, Nherf2-/- mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nherf2-/- mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta2-AR and CFTR was reduced in the Nherf1-/- mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Akt as a mediator of cell death

Protein kinase B/Akt possesses prosurvival and antiapoptotic activities and is involved in growth factor-mediated neuronal protection. In this study we establish Akt deactivation as a causal mediator of cell death. Akt deactivation occurs in multiple models of cell death including N-methyl-d-aspartate excitotoxicity, vascular stroke, and nitric oxide (NO)- and hydrogen peroxide (H2O2)-elicited death of HeLa, PC12, and Jurkat T cells. Akt deactivation characterizes both caspase-dependent and -independent cell death. Conditions rescuing cell death, such as treatment with poly(ADP-ribose) polymerase or NO synthase inhibitors and preconditioning with sublethal concentrations of N-methyl-d-aspartate, restore Akt activity. Infection of neurons with adenovirus expressing constitutively active Akt prevents excitotoxicity, whereas phosphatidylinositol 3-kinase inhibitors or infection with dominant negative Akt induce death of untreated neuronal cells.

Free radicals and other reactive oxygen metabolites in inflammatory bowel disease: cause, consequence or epiphenomenon?

Oxygen-derived free radicals and other reactive oxygen metabolites have emerged as a common pathway of tissue injury in a wide variety of otherwise disparate disease processes. This has given rise to the hope that efforts directed towards the pharmacologic control of free radical-mediated tissue injury (Reilly, P.M., Schiller, H. J. and Bulkley, G. B. (1991) Pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites. Am. J. Surg. 161: 488-503) may have particular application to patients suffering from Crohns disease and/or ulcerative colitis. However, because tissue injury by any mechanism, even direct mechanical trauma, can elicit an inflammatory response which entails the secondary generation of toxic oxidants by neutrophils and tissue macrophages, it is important that the evidence for this association be examined critically, so as to discriminate the possibility of an etiologic role for these toxic compounds from their presence as a reflection of injury caused primarily by other agents. Similarly, in considering the therapeutic potential of free radical ablation for the treatment of patients with IBD it is important to distinguish between interventions that might specifically block the fundamental injury mechanism from those which would act in a more nonspecific, anti-inflammatory role.

NHERF family and NHE3 regulation

The intestinal and renal proximal tubule brush border (BB) Na+–H+ exchanger NHE3 binds to members of the NHERF (Na+–H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3‐containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.

POLARIZED DISTRIBUTION OF KEY MEMBRANE TRANSPORT PROTEINS IN THE RAT SUBMANDIBULAR GLAND

Abstract Immunofluorescence labelling and confocal microscopy were employed to examine the polarized distribution of several membrane transport proteins believed to be essential for salivary secretion in the rat submandibular gland. The Na+/K+-ATPase, Na+/H+ exchanger isoform 1 (NHE1), and the secretory Na+/K+/2Cl–cotransporter isoform were all found in the basolateral membranes of acinar and intralobular duct cells. Anion exchanger isoform 2 (AE2) was found only in the basolateral membranes of acinar cells, while AE1 was absent from glandular epithelial cells. Aquaporin 5 was detected in the apical membranes of acinar cells, while the cystic fibrosis transmembrane conductance regulator was found only in apical membranes of intralobular duct cells. NHEs 2 and 3 were found in the apical membranes of both acinar and intralobular duct cells. Our results are generally consistent with the expected distribution of most transporters based on previous physiological and pharmacological experiments. However, the apical localization of NHEs 2 and 3, and the presence of the secretory isoform of the Na+/K+/2Cl–cotransporter in intralobular duct cells were not predicted.

MOLECULAR CLONING AND EXPRESSION OF A CDNA ENCODING THE RABBIT ILEAL VILLUS CELL BASOLATERAL MEMBRANE NA+/H+ EXCHANGER

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′‐untranslated region, 2451 bp of open reading frame and 1163 bp of 3′‐untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8‐Br‐cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29‐18 and Caco‐2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta‐galactosidase and the C‐terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI‐AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the ‘housekeeping’ Na+/H+ exchanger.

Subcellular Redistribution Is Involved in Acute Regulation of the Brush Border Na+/H+ Exchanger Isoform 3 in Human Colon Adenocarcinoma Cell Line Caco-2 PROTEIN KINASE C-MEDIATED INHIBITION OF THE EXCHANGER

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in V max, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from ∼4.3 to ∼2.4. This translocation resulted in 10–15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes ∼50% to the overall protein kinase C-induced inhibition of the exchanger.

Structure/function studies of the epithelial isoforms of the mammalian Na+/H+ exchanger gene family

Na +/H + exchangers or antiporters are plasma membrane transport proteins, which in eukaryotes exchange extracellular Na + for intracellular H + with a stoichiometry of 1 : 1 [31,56]. In intact cells, Na + enters down the Na-K-ATPase generated electrochemical Na + gradient. All eukaryotic cells studied have plasma membrane Na +/H + exchangers, including yeast, Caenorhabditis elegans and crustaceans [1, 37, 49]. Prokaryotes have functionally similar Na +/H + exchanger proteins which regulate the intracellular Na + ion concentration and pH [38, 60]. In contrast to eukaryotic Na+/H + exchangers, prokaryotic Na+/H + exchangers are electrogenic, exchanging two intracellular Na + for 1 H + ; usually utilizing the intracellular H + ion electromotive force. In eukaryotic cells, the plasma membrane Na+/H + exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na + absorption [reviewed in 31]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a C1-/HCO3 exchanger, a NaHCO3 co-transporter, a Na+-dependent CI-/HCO3 exchanger and multiple mechanisms of H + extrusion [reviewed in 41], including the H-KATPase pump. In this review, we will focus on recent advances