CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca2+ dependence of ClC-Kb channels.

ClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50 %. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome.

Mutations in SLC2A2 gene reveal hGLUT2 function in pancreatic ß cell development.

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and ß cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced ß cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic ß cell development and insulin secretion.

Characterization of the mouse ClC-K1/Barttin chloride channel.

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression.

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance.

Heterogeneity in the processing of CLCN5 mutants related to Dent disease.

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease.

KCNQ1 K+ channels are involved in lipopolysaccharide-induced apoptosis of distal kidney cells.

Most bacteria initiate host inflammatory responses through interactions with epithelial cells. Lipopolysaccharide (LPS), a component of the bacterial cell wall is a major cause of septic shock in emergency care units and in the pathogenesis of acute renal failure. Kidney cells exposed to LPS undergo apoptotic changes, including cell volume decrease, phosphatidylserine exposure, caspase-3- and membrane K+ conductance -activation. Whole-cell configuration was used to identify K+ channels in primary and immortalized culture of mice distal convoluted tubules. LPS exposure induced a 3 fold increase in intracellular cAMP concentration and the activation of an outwardly rectifying K+ conductance in both immortalized and primary culture of distal cells. This LPS-induced current exhibited KCNQ1 K+ channel characteristics, i.e. inhibition by quinidine, chromanol293B and low dose of HMR1556 (IC50<1 microM) and insensitive to TEA and charybdotoxin. The background-like biophysical properties of the current suggest that the KCNQ1 pore-forming subunit is associated with a KCNE2 or KCNE3 ancillary subunit. RT-PCR experiments confirmed the presence of KCNQ1 and KCNE3 mRNA transcripts in primary culture of distal segments. Activation of the KCNQ1/KCNE3 K+ current appeared to be an essential step in the LPS-induced apoptosis process since HMR1556 blocked the LPS-induced- cell volume decrease, -caspase-3 activation and -phosphatidylserine exposure.

CFTR mediates apoptotic volume decrease and cell death by controlling glutathione efflux and ROS production in cultured mice proximal tubules.

We have previously shown that despite the presence of mRNA encoding CFTR, renal proximal cells do not exhibit cAMP-sensitive Cl(-) conductance (Rubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P. Am J Physiol Renal Physiol 275: F651-F663, 1998). Nevertheless, in these cells, CFTR plays a crucial role in the control of the volume-sensitive outwardly rectifying (VSOR) activated Cl(-) currents during hypotonic shock. The aim of this study was to determine the role of CFTR in the regulation of apoptosis volume decrease (AVD) and the apoptosis phenomenon. For this purpose, renal cells were immortalized from primary cultures of proximal convoluted tubules from cftr(+/+) and cftr(-/-) mice. Apoptosis was induced by staurosporine (STS; 1 microM). Cell volume, Cl(-) conductance, caspase-3 activity, intracellular level of reactive oxygen species (ROS), and glutathione content (GSH/GSSG) were monitored during AVD. In cftr(+/+) cells, AVD and caspase-3 activation were strongly impaired by conventional Cl(-) channel blockers and by a specific CFTR inhibitor (CFTR(inh)-172; 5 microM). STS induced activation of CFTR conductance within 15 min, which was progressively replaced by VSOR Cl(-) currents after 60 min of exposure. In parallel, STS induced an increase in ROS content in the time course of VSOR Cl(-) current activation. This increase was impaired by CFTR(inh)-172 and was not observed in cftr(-/-) cells. Furthermore, the intracellular GSH/GSSG content decreased during STS exposure in cftr(+/+) cells only. In conclusion, CFTR could play a key role in the cascade of events leading to apoptosis. This role probably involves control of the intracellular ROS balance by some CFTR-dependent modulation of GSH concentration.

Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules and is frequently associated with mutations in CLCN5, which encodes the electrogenic chloride-proton exchanger ClC-5. To better understand the functional consequences of CLCN5 mutations in this disease, we screened four newly identified missense mutations (G179D, S203L, G212A, L469P), one new nonsense mutation (R718X), and three known mutations (L200R, C219R, and C221R), in Xenopus laevis oocytes and HEK293 cells expressing either wild-type or mutant exchanger. A type-I mutant (G212A) trafficked normally to the cell surface and to early endosomes, underwent complex glycosylation at the cell surface like wild-type ClC-5, but exhibited significant reductions in outwardly rectifying ion currents. The type-II mutants (G179D, L200R, S203L, C219R, C221R, L469P, and R718X) were improperly N-glycosylated and were non-functional due to retention in the endoplasmic reticulum. Thus these mutations have distinct mechanisms by which they could impair ClC-5 function in Dent's disease.

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells.

The aim of this study was to characterize the role of CFTR during Cd(2+)-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr(+/+) and cftr(-/-) mice were used. In cftr(+/+) cells, the application of Cd(2+) (5 microM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl(-) conductance sensitive to CFTR(inh)-172. Thereafter Cd(2+) induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl(-) and TASK2 K(+) conductances. By contrast, cftr(-/-) cells exposed to Cd(2+) were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr(+/+) cells, Cd(2+) enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR(inh)-172 or in cftr(-/-) cells. Additionally, Cd(2+) exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr(+/+) cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd(2+) exposure by modulating the intracellular content of glutathione.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Role of TASK2 in the control of apoptotic volume decrease in proximal kidney cells.

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.

Cancer cell cycle modulated by a functional coupling between sigma-1 receptors and Cl- channels.

The sigma-1 receptor is an intracellular protein characterized as a tumor biomarker whose function remains mysterious. We demonstrate herein for the first time that highly selective sigma ligands inhibit volume-regulated chloride channels (VRCC) in small cell lung cancer and T-leukemia cells. Sigma ligands and VRCC blockers provoked a cell cycle arrest underlined by p27 accumulation. In stably sigma-1 receptor-transfected HEK cells, the proliferation rate was significantly lowered by sigma ligands when compared with control cells. Sigma ligands produced a strong inhibition of VRCC in HEK-transfected cells but not in control HEK. Surprisingly, the activation rate of VRCC was dramatically delayed in HEK-transfected cells in the absence of ligands, indicating that sigma-1 receptors per se modulate cell regulating volume processes in physiological conditions. Volume measurements in hypotonic conditions revealed indeed that the regulatory volume decrease was delayed in HEK-transfected cells and virtually abolished in the presence of igmesine in both HEK-transfected and T-leukemic cells. Moreover, HEK-transfected cells showed a significant resistance to staurosporine-induced apoptosis volume decrease, indicating that sigma-1 receptors protect cancer cells from apoptosis. Altogether, our results show for the first time that sigma-1 receptors modulate "cell destiny" through VRCC and cell volume regulation.

AIP4 restricts transforming growth factor-beta signaling through a ubiquitination-independent mechanism.

Smad7 functions as an intracellular antagonist in transforming growth factor-beta (TGF-beta) signaling. In addition to interacting stably with the activated TGF-beta type I receptor (TbetaRI) to prevent phosphorylation of the receptor-regulated Smads (Smad2 and Smad3), Smad7 also induces degradation of the activated TbetaRI through association with different E3 ubiquitin ligases. Using the two-hybrid screen, we identified atrophin 1-interacting protein 4 (AIP4) as an E3 ubiquitin ligase that specifically targets Smad7 for ubiquitin-dependent degradation without affecting the turnover of the activated TbetaRI. Surprisingly, we found that despite the ability to degrade Smad7, AIP4 can inhibit TGF-beta signaling, presumably by enhancing the association of Smad7 with the activated TbetaRI. Consistent with this notion, expression of a catalytic mutant of AIP4, which is unable to induce ubiquitination and degradation of Smad7, also stabilizes the TbetaRI.Smad7 complex, resulting in inhibition of TGF-beta signaling. The ability of AIP4 to enhance the inhibitory function of Smad7 independent of its ubiquitin ligase activity reveals a new mechanism by which E3 ubiquitin ligases may function to turn off TGF-beta signaling.

Interaction of pyrin with 14.3.3 in an isoform-specific and phosphorylation-dependent manner regulates its translocation to the nucleus.

OBJECTIVE: Pyrin, the familial Mediterranean fever gene product, exists in several isoforms of unknown functions. The recombinant full-length isoform (pyrin.fl) is cytoplasmic, whereas an alternatively spliced isoform lacking exon 2 (pyrin.DeltaEx2) concentrates in the nucleus. Native pyrin, mainly consisting of pyrin.fl, is also cytoplasmic in monocytes but is predominantly nuclear in other cell types. To understand pyrin-dependent biologic pathways and to decipher the mechanisms accounting for such different patterns of subcellular compartmentalization, binding partners and posttranslational modifications of pyrin were assessed. METHODS: A yeast 2-hybrid screen was performed with pyrin.fl as the bait. The interaction identified between pyrin.fl and 14.3.3 proteins was confirmed by immunoprecipitation of (35)S-radiolabeled protein complexes; similar experiments were performed with pyrin.DeltaEx2, pyrin.fl after alkaline phosphatase treatment, and pyrin.fl mutants in which several exon 2-encoded serine residues were replaced by nonphosphorylatable alanines. The subcellular localization of the different wild-type and mutated pyrin proteins was assessed by immunofluorescence. RESULTS: Two members of the 14.3.3 protein family were identified as pyrin partners. Whereas pyrin.fl interacted with 14.3.3tau and 14.3.3epsilon, these interactions did not occur with pyrin.DeltaEx2. Pyrin.fl was phosphorylated, and this modification mediated 14.3.3 binding. Serines 208, 209, and 242, within exon 2, acted as critical residues in the interaction between pyrin.fl and 14.3.3. When an S208-S209-S242A pyrin.fl triple mutant or wild-type pyrin.fl in the presence of an inhibitor of 14.3.3-ligand interactions was used, promotion of nuclear translocation of pyrin was observed. CONCLUSION: These results disclose the role played by 14.3.3 in the regulation of the subcellular compartmentalization of pyrin in a phosphorylation- and isoform-dependent manner. They also reconcile the observations made in vitro with those made in vivo, while providing a direct link between 14.3.3-dependent pathways and pyrin.

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac.

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.

The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation.

Ubiquitin-dependent degradation plays an important role in the negative regulation of TGF-beta signaling. Here, we identify Tiul1 (for TGIF interacting ubiquitin ligase 1), a novel E3 ubiquitin ligase that inhibits TGF-beta signaling by targeting both the activated receptor and Smad2 for degradation. Tiul1 associates constitutively with Smad7 and induces degradation of the activated type I receptor without affecting the expression levels of Smad7. Tiul1 can also interact with Smad2 and the nuclear corepressor TGIF upon activation of TGF-beta signaling. Like Smad7, the steady-state levels of TGIF are not affected by Tiul1, but the interaction of Tiul1 with TGIF allows this ubiquitin ligase to target Smad2 for degradation. Consistent with this, overexpression of Tiul1 suppressed TGF-beta-induced growth arrest and transcriptional responses. In addition, silencing of Tiul1 or TGIF genes by siRNA resulted in suppression of the TGF-beta-dependent degradation of Smad2 and an enhancement of TGF-beta-mediated gene expression. These results reveal a new role for TGIF as a component of a ubiquitin ligase complex that mediates the degradation of Smad2 in response to TGF-beta signaling.

RLIP, an effector of the Ral GTPases, is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis.

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. We have looked for actors that associate with RLIP during mitosis. We show here that RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and that this interaction is crucial for the mitotic phosphorylation of Epsin that, once phosphorylated, is no longer competent for endocytosis. We show also that this latter phosphorylation is dependent on Ral signaling. We propose that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off.

A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways.

Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities.

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling.

Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling.

Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins.

The GTPase-activating protein RasGAP functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. As RasGAP itself has no obvious enzymatic function that may explain this effector function, we looked for downstream RasGAP effectors that could fulfill this role. We looked for the existence of RasGAP Src homology 3 (SH3) domain partners as this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. We report here the identification of a new RasGAP SH3 domain-binding protein, named Aurora. This Drosophila melanogaster Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2, and -3. Coimmunoprecipitation experiments in COS cells confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed that it interacts with HsAIRK-1 in G(2)/M HeLa cells. We also demonstrated that RasGAP binds to the kinase domain of Aurora and that this interaction inhibits the kinase activity of HsAIRK-1 and HsAIRK-2. Finally we showed that RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis.