Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation.

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca(2+) uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca(2+)-dependent ROS production.

Electrogenic bicarbonate secretion by prairie dog gallbladder.

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (I(sc); 171 +/- 21 microA/cm(2)) and lumen-negative potential difference (-10.1 +/- 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to I(sc). In response to 2 microM forskolin, I(sc) exceeded 270 microA/cm(2), and impedance estimates of the apical membrane resistance decreased from 200 Omega.cm(2) to 13 Omega.cm(2). The forskolin-induced I(sc) was dependent on extracellular HCO(3)(-) and was blocked by serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl(-) ion substitution had little effect. Serosal trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba(2+) reduced I(sc), consistent with the inhibition of cAMP-dependent K(+) channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO(3)(-) entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K(+) channel activation.

Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway.

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, (1424)YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the mu subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of mu 2. Furthermore, isolated mu 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that mu 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative mu 2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the mu 2 subunit of AP-2 and mutations in mu 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling.

Endocytic adaptor complexes bind the C-terminal domain of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions at the apical membrane of epithelial cells to regulate chloride permeability. Recent studies have shown that CFTR is rapidly and efficiently internalized from the plasma membrane. We have shown that such internalization is mediated solely by clathrin-coated pathways, and that other pathways, such as caveolae, exclude CFTR. Moreover, CFTR co-precipitates with alpha-adaptin, a component of the endocytic adaptor complex (AP-2). The goal of our current studies was to elucidate further the molecular mechanisms that facilitate entry of CFTR into endocytic clathrin-coated vesicles. Protein-protein interactions generated by incubation of full-length in-vitro-translated CFTR with partially purified bovine brain adaptor complexes were evaluated following immunoprecipitation using an antibody against the alpha-adaptin subunit of the AP-2 complex. Such studies revealed co-immunoprecipitation of alpha-adaptin with full-length but not partially translated CFTR, suggesting that the C-terminus of CFTR may be responsible for this interaction. To test this hypothesis a C-terminal GST fusion protein (amino acids 1404-1480; CF-GST) was used in a "pull-down" assay with purified adaptor complexes. CF-GST sepharose was able to pull-down AP-2 endocytic adaptor complexes, as determined by immunoblot analyses of the precipitates using antibodies directed against alpha-adaptin. In contrast, CF-GST sepharose was unable to pull-down gamma-adaptin, a component of the Golgi-derived AP-1 clathrin adaptor complex. Thus, we demonstrate that CFTR is endocytosed via clathrin-coated vesicles, and that targeting of CFTR to these structures is mediated by binding of the AP-2 adaptor complex to the C-terminal domain of CFTR.

cAMP signaling cascades and CFTR: is there more to learn?

CFTR is an apically resident ion channel whose activity is regulated by the activation of the cAMP mediated second messenger cascade. As depicted in textbooks, the cAMP mediated signaling cascade appears deceptively simple, yet, our growing understanding of this pathway shows it to be much more complicated and finely tuned than originally thought. The intent of this review is to evaluate our current understanding of the cAMP mediated signaling as it relates to the secretion of mucin and chloride, two compounds whose regulated secretion is altered in cystic fibrosis.

Protein kinase-A-mediated secretion of mucin from human colonic epithelial cells.

Both Ca(2+)- and cAMP-mediated second messenger cascades acutely regulate mucin secretion from human colonic epithelial cells. To better understand the cAMP-dependent regulation of mucin secretion we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms in mucus-secreting T84 cells, and determined which of these isoforms is responsible for agonist-stimulated mucin secretion. Our results show the presence of both type I and type II PKA in cells that also contain large mucin granules. Forskolin caused a rapid and sustained increase in PKA activity that reached a maximum 5-10 min following its addition. Secretion of mucin was detected 15 min following exposure to forskolin, and continued to increase for a further 15 min before reaching a plateau. Mucin secretion was also measured in the presence of combinations of site-selective cAMP analog pairs, which preferentially activate either type I or type II PKA. Similar levels of mucin secretion were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase mucin secretion. Thus, activation of either type I or type II PKA is able to maximally stimulate secretion of mucins from T84 human colonic epithelial cells.

Estrogen inhibition of cystic fibrosis transmembrane conductance regulator-mediated chloride secretion.

Cystic fibrosis (CF) is an autosomal genetic disease associated with impaired epithelial ion transport. Mutations in the CF gene alter the primary sequence of the CF transmembrane conductance regulator (CFTR). Several therapeutic modalities have been proposed for CF patients, including the phytoestrogen genistein. Experiments were completed in cellular and subcellular systems to evaluate the impact of naturally occurring and synthetic estrogens on epithelial ion transport, and specifically on the CF protein CFTR. 17beta-Estradiol, a naturally occurring estrogen, caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers with a K(i) of 8 microM. In addition, 17alpha-estradiol, a stereoisomer that fails to bind and activate nuclear estrogen receptors was equipotent with 17beta-estradiol, arguing against a genomic-mediated mechanism of action. Synthetic estrogens, including diethylstilbesterol and the antiestrogen tamoxifen likewise inhibited forskolin-stimulated ion transport. Aldosterone, dexamethasone, and cholesterol were without effect at the highest concentrations tested (>/=1 mM). Studies indicated that diethylstilbesterol and other synthetic estrogens that inhibited anion secretion in intact monolayers likewise inhibited CFTR chloride channel activity with similar concentration dependencies in excised membrane patches. Experiments with radioactive photoactivatable estrogen derivatives demonstrated that these compounds bind directly to CFTR expressed in insect cells. Taken together, the data suggest that estrogens can interact directly with CFTR to alter anion transport.

E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.

The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors.

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid and efficient endocytosis. Since functionally active CFTR is found in purified clathrin-coated vesicles isolated from both cultured epithelial cells and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivo cross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carboxyl terminus of CFTR (amino acids 1404-1480) were also able to bind AP-2 but did not bind the Golgi-specific adaptor complex AP-1. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Secondary structure predictions suggest that Tyr(1424) is present in a beta-turn conformation, a conformation disrupted by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate efficient entry of CFTR into clathrin-coated vesicles.

Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.

Intracellular CFTR: localization and function.

Intracellular CFTR: Localization and Function. Physiol. Rev. 79, Suppl.: S175-S191, 1999. - There is considerable evidence that CFTR can function as a chloride-selective anion channel. Moreover, this function has been localized to the apical membrane of chloride secretory epithelial cells. However, because cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein, it will also be present, to some degree, in a variety of other membrane compartments (including endoplasmic reticulum, Golgi stacks, endosomes, and lysosomes). An incomplete understanding of the molecular mechanisms by which alterations in an apical membrane chloride conductance could give rise to the various clinical manifestations of cystic fibrosis has prompted the suggestion that CFTR may also play a role in the normal function of certain intracellular compartments. A variety of intracellular functions have been attributed to CFTR, including regulation of membrane vesicle trafficking and fusion, acidification of organelles, and transport of small anions. This paper aims to review the evidence for localization of CFTR in intracellular organelles and the potential physiological consequences of that localization.

Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells.

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIalpha and RIIalpha. In addition, immunogold electron microscopy showed that RIIalpha labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIalpha was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Calpha was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (approximately 100 microA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.

Role of membrane trafficking in plasma membrane solute transport.

Cells can rapidly and reversibly alter solute transport rates by changing the kinetics of transport proteins resident within the plasma membrane. Most notably, this can be brought about by reversible phosphorylation of the transporter. An additional mechanism for acute regulation of plasma membrane transport rates is by the regulated exocytic insertion of transport proteins from intracellular vesicles into the plasma membrane and their subsequent regulated endocytic retrieval. Over the past few years, the number of transporters undergoing this regulated trafficking has increased dramatically, such that what was once an interesting translocation of a few transporters has now become a widespread modality for regulating plasma membrane solute permeabilities. The aim of this article is to review the models proposed for the regulated trafficking of transport proteins and what lines of evidence should be obtained to document regulated exocytic insertion and endocytic retrieval of transport proteins. We highlight four transporters, the insulin-responsive glucose transporter, the antidiuretic hormone-responsive water channel, the urinary bladder H(+)-ATPase, and the cystic fibrosis transmembrane conductance regulator Cl- channel, and discuss the various approaches taken to document their regulated trafficking. Finally, we discuss areas of uncertainty that remain to be investigated concerning the molecular mechanisms involved in regulating the trafficking of proteins.

Biochemical and biophysical identification of cystic fibrosis transmembrane conductance regulator chloride channels as components of endocytic clathrin-coated vesicles.

Cystic fibrosis results from mutations in the gene encoding the CFTR Cl- channel. Although CFTR occurs as an integral component of the plasma membrane, recent studies implicate CFTR in endocytic recycling and suggest that the protein may also exist in intracellular vesicular compartments. To test this, we analyzed CFTR in clathrin-coated vesicles (CCV) purified from cells constitutively expressing CFTR at high levels. CFTR immunoreactivity was detected in CCV by immunoblot and was identified as CFTR based on labeling of immunoprecipitates with protein kinase A and by tryptic phosphopeptide mapping. Fusion of uncoated CCV with planar lipid bilayers resulted in the incorporation of kinase- and ATP-activated Cl- channel activity (7.8 pS at 20 degrees C; 11.9 pS at 37 degrees C), with a linear current-voltage relation under symmetrical conditions. Thus, functional CFTR occurs in CCV. Moreover, CFTR interacts with the plasma membrane specific adaptor complex during endocytosis through clathrin-coated pits. Therefore, the abundance of CFTR in the plasma membrane may be regulated by exocytic insertion and endocytic recycling, and these processes may provide an augmentation to protein kinase A activation as a mechanism for regulating CFTR Cl channels in the plasma membrane.

Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line.

Endocytosis in the chloride secreting epithelial cell line T84 was monitored by uptake of the fluid-phase markers FITC-dextran and horseradish peroxidase (HRP). Uptake of marker was inhibited by incubation of cells at 4 degrees C, consistent with an endocytic uptake. Although activation of the cAMP-dependent second messenger pathway has been shown to stimulate exocytosis in this cell line, it caused a 63% reduction in endocytosis as measured by uptake of fluid-phase markers. In contrast, the presence of the protein kinase C activator phorbol-myristate acetate (PMA) caused no significant reduction in the level of endocytosis compared to control, nor did it reverse the inhibitory effect of PKA activation. The data thus suggest that endocytosis in T84 cells is regulated through activation of protein kinase A, but not through activation of protein kinase C.

Regulation of plasma membrane recycling by CFTR.

The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.