Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the Fc?RI Pathway in Mast Cells

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells.Mast cells reside in connective and mucosal tissues and play a key protective role in the immune response to helminth infection (13, 21), sepsis (39), and snake or bee venoms (42). Mast cells express FcɛRI, which becomes sensitized to antigens or allergens upon immunoglobulin E (IgE) binding. Aggregation of FcɛRI by multivalent antigens causes the release of preformed mediators by degranulation and the de novo production of lipid mediators and cytokines (1, 52). Release of these mediators causes increased vascular permeability, leukocyte recruitment and activation, and inflammation (41). Aberrant mast cell activation is implicated in IgE-mediated type I hypersensitivity reactions including anaphylaxis, allergic rhinitis, and asthma (20). FcɛRI is a tetrameric receptor composed of an IgE-binding α chain and of β and γ chains containing immunoreceptor tyrosine-based activation motifs that become phosphorylated following multivalent antigen-mediated clustering of FcɛRI and activation of Src family protein tyrosine kinases (PTKs), primarily involving Lyn (51). Lyn phosphorylates and activates both positive effectors of FcɛRI signaling (e.g., Syk PTK) and key negative regulators (e.g., Shp-1 and SHIP) that serve to limit mast cell activation (28, 46, 69).Fes (the mammalian orthologue of the v-Fps and v-Fes oncoproteins from avian [57, 58] and feline [15, 56] retroviruses) and Fer are closely related PTKs that become activated following FcɛRI aggregation in mast cells (10). Surprisingly, FcɛRI-induced tyrosine phosphorylation of Fes and Fer does not require their kinase activities (55) and is almost entirely dependent on Lyn (67). Through the use of transgenic mouse models, evidence for both unique and redundant functions for Fes and Fer has been described in regulating hematopoiesis (55) and limiting the innate immune response (22, 40, 50, 72). In mast cells, we have shown that Fer promotes activation of p38 mitogen-activated protein kinase and chemotaxis of mast cells (10). We also found that Fer and Fes PTKs contribute to FcɛRI-evoked phosphorylation of platelet-endothelial cell adhesion molecule 1 (PECAM-1) (67).Each of the Fes and Fer PTKs is composed of an N-terminal regulatory domain containing a Fer-CIP4 homology (FCH) domain followed by several predicted coiled-coils (CC), a central SH2 domain, and C-terminal PTK domain (19). It is worth noting that early studies pointed toward an important role for the N-terminal domain of v-Fps for its transforming activity and membrane localization (5, 63). Several recent studies have defined the FCH and first CC domain (amino acids 1 to 300) in Fer, CIP4, and other pombe Cdc15 homology (PCH) family adaptor proteins as an F-BAR domain (also termed extended FCH or EFC domain) (reviewed in references 3 and 9). The F-BAR domain was found to constitute a novel phosphoinositide-binding domain that can promote tubulation of liposomes in vitro and membranes in vivo (27, 33, 66). The crystal structures of F-BAR domains from several PCH adaptors were recently solved (27, 59). The F-BAR module was shown to consist of a triple helical bundle that forms a homodimer, with a concave surface rich in basic residues that have recently been shown to contact phospholipids in curved membranes (16). In vitro studies using the Fer F-BAR domain have shown that the F-BAR domain binds strongly to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]; however, the Fer F-BAR is relatively weak compared with the adaptor protein FBP17 at inducing membrane tubulation (66). Liposome sedimentation assays have identified several conserved basic residues required for F-BAR domain binding to PI(4,5)P₂(66). The substitution of R113/K114 to glutamines (RK/QQ) in FBP17 reduced phosphoinositide binding by 80% (66). A recent electron cryoelectron microscopy study provided insights into binding of F-BAR dimers to flat and curved membranes via different binding faces (16). This study also confirmed that R113/K114 residues (in CIP4) constitute a site of direct interaction with the liposomes. Interestingly, microdomains of the plasma membrane rich in PI(4,5)P₂ are sites of dynamic actin assembly (47) and endocytosis (4, 31). Previous studies have described Fes localization to a variety of subcellular structures, including endocytic vesicles (71), the trans-Golgi apparatus (71), microtubules (37), and focal adhesions (44). The rapid activation of Fes and Fer PTKs upon FcɛRI aggregation on mast cells (10) would suggest that there is a mechanism by which Fes localizes at or near the plasma membrane. Phosphoinositide-binding via the F-BAR domain of Fes and Fer PTKs may promote their recruitment to the plasma membrane prior to their activation by cell surface receptors such as FcɛRI. The potential colocalization with endocytosis and actin assembly regulators may allow for regulation of receptor endocytosis or chemotaxis of mast cells by Fes/Fer PTKs. A recent study implicates Rab5 GTPase and its exchange factor RabGEF1/Rabex-5 in promoting internalization of FcɛRI following clustering by antigens (34). It is worth noting that defects in internalization of Toll-like receptor 4 and transferrin receptor were observed in Fes-deficient macrophages (48), and there is a potential role for Fes in regulating internalization of mast cell receptors.In this study, we provide novel insights into the phospholipid binding and liposome tubulating properties of the Fes F-BAR domain. Mutation of two conserved basic residues within the Fes F-BAR domain (RK/QQ) reduced phospholipid binding in vitro, and membrane localization in vivo. In transfected RBL-2H3 mast cells, the Fes harboring the RK/QQ mutation (Fes^RK/QQ) displayed reduced FcɛRI-evoked tyrosine phosphorylation compared to wild-type Fes (Fes^WT), which correlated with reduced localization to Lyn-containing membranes in mast cells. The SH2 domain of Fes was found to interact with several phosphoproteins in mast cells, including FcɛRI and HS1, an actin regulator and cortactin homologue. We found that Fes contributes to HS1 phosphorylation at C-terminal residues implicated in actin branch stabilization, and we present a model for how F-BAR-containing adaptor proteins and PTKs may coordinate actin-driven endocytosis in mast cells.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

The Influence of Fluoride on the Expression of Inhibitors of Wnt/β-Catenin Signaling Pathway in Rat Skin Fibroblast Cells

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/β-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST),were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls(0 mg F(?)/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/β-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.     

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).

Methodology/Principal Findings

VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38^MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.

Conclusions/Significance

In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38^MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.     

Increasing the Receptor Tyrosine Kinase EphB2 Prevents Amyloid-β-induced Depletion of Cell Surface Glutamate Receptors by a Mechanism That Requires the PDZ-binding Motif of EphB2 and Neuronal Activity

Diverse lines of evidence suggest that amyloid-β (Aβ) peptides causally contribute to the pathogenesis of Alzheimer disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wild-type or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity but not on its kinase activity. We further present evidence that the protective effect of EphB2 may be mediated by the AMPA-type glutamate receptor subunit GluA2, which can become associated with the PDZ-binding motif of EphB2 through PDZ domain-containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrP^C), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPA-type glutamate receptors.     

Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II

We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.     

The Daf2-2 Mutation, a Dominant Inhibitor of the Ste4 Step in the α-Factor Signaling Pathway of Saccharomyces Cerevisiae Matα Cells

A dominant mutation (DAF2-2) resulting in resistance to the mating pheromone alpha-factor in Saccharomyces cerevisiae MATa cells was identified and characterized genetically. Whereas wild-type cells induce a high level of the FUS1 mRNA from a low baseline on exposure to alpha-factor, DAF2-2 cells were constitutive producers of an intermediate level of FUS1 RNA; the level was increased only modestly by alpha-factor. FUS1 constitutivity required STE4, STE5 and STE18, but did not require STE2, the alpha-factor receptor gene. DAF2-2 suppressed the alpha-factor supersensitivity of a STE2 C-terminal truncation, and suppressed lethality due to scg1 mutations. Thus DAF2-2 may act by uncoupling the signaling pathway from alpha-factor binding at some point in the pathway between Scg1 inactivation and the action of Ste4, Ste5 and Ste18; this uncoupling might occur at the expense of partial constitutive activation of the pathway. DAF2-2 suppressed the unconditional cell-cycle arrest phenotype of a dominant "constitutive signaling" allele of STE4 (STE4Hpl), although the constitutive FUS1 phenotype of DAF2-2 was suppressed by ste4 null mutations; therefore DAF2-2 may directly affect the performance of the STE4 step.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

Protein Kinase C�� Supports Survival of MDA-MB-231 Breast Cancer Cells by Suppressing the ERK1/2 Pathway

Mechanisms that mediate apoptosis resistance are attractive therapeutic targets for cancer. Protein kinase Cδ (PKCδ) is considered a pro-apoptotic factor in many cell types. In breast cancer, however, it has shown both pro-survival and pro-apoptotic effects. Here, we report for the first time that down-regulation of PKCδ per se leads to apoptosis of MDA-MB-231 cells. Inhibition of MEK1/2 by either PD98059 or U0126 suppressed the induction of apoptosis of PKCδ-depleted MDA-MB-231 cells but did not support survival of MCF-7 or MDA-MB-468 cells. Basal ERK1/2 phosphorylation was substantially higher in MDA-MB-231 cells than in the other cell lines. PKCδ depletion led to even higher ERK1/2 phosphorylation levels and also to lower expression levels of the ERK1/2 phosphatase MKP3. Depletion of MKP3 led to apoptosis and higher levels of ERK1/2 phosphorylation, suggesting that this may be a mechanism mediating the effect of PKCδ down-regulation. However, PKCδ silencing also induced increased MEK1/2 phosphorylation, indicating that PKCδ regulates ERK1/2 phosphorylation both upstream and downstream. Moreover, PKCδ silencing led to increased levels of the E3 ubiquitin ligase Nedd4, which is a potential regulator of MKP3, because down-regulation led to increased MKP3 levels. Our results highlight PKCδ as a potential target for therapy of breast cancers with high activity of the ERK1/2 pathway.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Gastric Cancer Exosomes Trigger Differentiation of Umbilical Cord Derived Mesenchymal Stem Cells to Carcinoma-Associated Fibroblasts through TGF-β/Smad Pathway

Background

Mesenchymal stem cells (MSCs) promote tumor growth by differentiating into carcinoma-associated fibroblasts (CAFs) and composing the tumor microenvironment. However, the mechanisms responsible for the transition of MSCs to CAFs are not well understood. Exosomes regulate cellular activities by mediating cell-cell communication. In this study, we aimed to investigate whether cancer cell-derived exosomes were involved in regulating the differentiation of human umbilical cord-derived MSCs (hucMSCs) to CAFs.

Methodology/Principal Findings

We first showed that gastric cancer cell-derived exosomes induced the expression of CAF markers in hucMSCs. We then demonstrated that gastric cancer cell-derived exosomes stimulated the phosphorylation of Smad-2 in hucMSCs. We further confirmed that TGF-β receptor 1 kinase inhibitor attenuated Smad-2 phosphorylation and CAF marker expression in hucMSCs after exposure to gastric cancer cell-derived exosomes.

Conclusion/Significance

Our results suggest that gastric cancer cells triggered the differentiation of hucMSCs to CAFs by exosomes-mediated TGF-β transfer and TGF-β/Smad pathway activation, which may represent a novel mechanism for MSCs to CAFs transition in cancer.     

Secreted Products of Macrophages Exposed to Calcium Oxalate Crystals Induce Epithelial Mesenchymal Transition of Renal Tubular Cells via RhoA-Dependent TGF-β1 Pathway

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999