Caveolin-1 function at the plasma membrane and in intracellular compartments in cancer.

Caveolin-1 (CAV1) is commonly considered to function as a cell surface protein, for instance in the genesis of caveolae. Nonetheless, it is also present in many intracellular organelles and compartments. The contributions of these intracellular pools to CAV1 function are generally less well understood, and this is also the case in the context of cancer. This review will summarize literature available on the role of CAV1 in cancer, highlighting particularly our understanding of the canonical (CAV1 in the plasma membrane) and non-canonical pathways (CAV1 in organelles and exosomes) linked to the dual role of the protein as a tumor suppressor and promoter of metastasis. With this in mind, we will focus on recently emerging concepts linking CAV1 function to the regulation of intracellular organelle communication within the same cell where CAV1 is expressed. However, we now know that CAV1 can be released from cells in exosomes and generate systemic effects. Thus, we will also elaborate on how CAV1 participates in intracellular communication between organelles as well as signaling between cells (non-canonical pathways) in cancer.

Hypoxia-Induced Caveolin-1 Expression Promotes Migration and Invasion of Tumor Cells.

BACKGROUND: Exacerbated proliferation of cancer cells in nascent tumors leads to the genesis of a hypoxic microenvironment, which is associated with poor patient prognosis, because these stress conditions enhance migratory, invasive and metastatic capacities of tumor cells. These changes are associated with the induction of the hypoxia-inducible factors (HIFs, mainly HIF1α) and increased expression of target genes, including Caveolin-1 (CAV1). Results from our group have shown that CAV1 expression in metastatic cancer cells promotes cell migration/invasion in vitro and metastasis in vivo in a manner dependent on tyrosine-14 phosphorylation by src family kinases. Here, we evaluated whether hypoxia-induced expression of CAV1 was required for hypoxia-dependent migration and invasion in cancer cells. METHODS: B16-F10 murine melanoma and HT29(US) colon adenocarcinoma cells were exposed to hypoxia (1% O2). CAV1 expression was evaluated by western blotting. Endogenous CAV1 and HIF1α were knocked-down using different shRNA constructs. Cell migration and invasion were evaluated in Boyden Chamber and Matrigel assays, respectively. RESULTS: We observed that hypoxia increased CAV1 protein levels in a HIF1 α- dependent manner, in B16-F10 and HT29(US) cells. Importantly, hypoxia-dependent migration of both tumor cell lines was blocked upon CAV1 knock-down. Likewise, pharmacological inhibition of HIF prevented hypoxia-induced migration and invasion in B16-F10 cells. Finally, hypoxia-induced migration was also blocked by the src-family kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo3,4-dpyrimidine (PP2), an inhibitor of CAV1 phosphorylation. CONCLUSION: Hypoxia induced migration and invasion of metastatic cancer cells require HIF1α-dependent induction of CAV1 expression and src family kinase activation.

The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.

The twisted survivin connection to angiogenesis.

Survivin, a member of the inhibitor of apoptosis family of proteins (IAPs) that controls cell division, apoptosis, metastasis and angiogenesis, is overexpressed in essentially all human cancers. As a consequence, the gene/protein is considered an attractive target for cancer treatment. Here, we discuss recent findings related to the regulation of survivin expression and its role in angiogenesis, particularly in the context of hypoxia. We propose a novel role for survivin in cancer, whereby expression of the protein in tumor cells promotes VEGF synthesis, secretion and angiogenesis. Mechanistically, we propose the existence of a positive feed-back loop involving PI3-kinase/Akt activation and enhanced ß-Catenin-TCF/LEF-dependent VEGF expression followed by secretion. Finally, we elaborate on the possibility that this mechanism operating in cancer cells may contribute to enhanced tumor vascularization by vasculogenic mimicry together with conventional angiogenesis.

Caveolin-1 in cell migration and metastasis.

Caveolin-1 is a member of the caveolin family that has been ascribed a dual role in cancer. In early stages of disease the protein functions predominantly as a tumor suppressor, whereas at later stages, caveolin-1 expression is associated with tumor progression and metastasis. Here, some mechanisms associated with caveolin-1-dependent tumor suppression will be briefly discussed before focusing on the role of this protein and particularly phosphorylation of tyrosine-14 in promoting cell migration, invasion and metastasis. Models are provided summarizing possible explanations for these dramatic changes in function, as well as mechanisms by which this may be achieved.

Testosterone modulates the expression of molecules linked to insulin action and glucose uptake in endometrial cells.

Polycystic ovary syndrome (PCOS) is a common hyperandrogenic disorder associated with insulin resistance. Insulin exerts its metabolic function by activating the PI3K/Akt pathway and favoring glucose uptake. Caveolin-1 is a scaffolding protein which increases insulin receptor (IR) stability. Alternatively, activation of IR increases caveolin-1 phosphorylation on tyrosine-14. Furthermore, endometrial tissue from PCOS patients is proposed to be insulin resistant; however, the particular role of testosterone in modulating the metabolic effects of insulin remains unexplored in endometrial stromal cells. To evaluate whether androgens modulate the response to insulin, T-HESCs cells were stimulated with 100 nM testosterone for 24 h and changes in the protein levels of caveolin-1, IR, and Akt were determined by Western blotting (WB). After testosterone treatment, the consequences of acute insulin stimulation were evaluated by WB analysis of phospho-S473Akt and phospho-Y14Caveolin-1, as well as by measuring glucose incorporation analyzing 2-deoxyglucose (2-DOG) uptake. For cells pretreated with testosterone, higher IR, IRS-1, and caveolin-1 protein levels compared with control conditions were detected. However, in testosterone treated cells acute insulin stimulation did not increase phospho-S473Akt and phospho-Y14caveolin-1 levels and reduced 2-DOG uptake was observed compared to control cells. Our results suggest that testosterone may have a detrimental role on the metabolic effects of insulin in endometrial stromal cell cultures. Thus, the high androgen levels in patients with PCOS may favor insulin resistance observed in endometria from these women.

Sjögren's syndrome and the epithelial target: a comprehensive review.

The most difficult component in our understanding of human autoimmunity remains a rigorous dissection of etiological events. Indeed, the vast literature on autoimmune diseases focuses on the inflammatory response, with the hope of developing drugs that reduce inflammation. However, there is increasing recognition that understanding the immunobiology of target tissues will also have direct relevance to disease natural history, including breach of tolerance. Sjögren's syndrome is essentially an epitheliitis and there are major changes to normal architectural salivary organization. We propose that loss of homeostasis is the initial event that precipitates inflammation and that such inflammatory response includes not only the adaptive response, but also an intense innate immune/bystander response. To understand these events this review focuses on the architecture, phenotype, function and epithelial cell organization. We further submit that there are several critical issues that must be defined to fully understand epithelial cell immunobiology in Sjögren's syndrome, including defining epithelial cell polarity, cell-cell and cell to extracellular matrix interactions and a variety of chemical and mechanical signals. We also argue that disruption of tight junctions induces disorganization of the apical pole of salivary acinar cells in Sjögren's syndrome. In addition, there will be a critical role of inflammatory cytokines in the apico-basal relocation of tight junction proteins. Further, the altered disorganization and relocation of proteins that participate in secretory granule formation are also dysregulated in Sjögren's syndrome and will contribute to abnormalities of mucins within the extracellular matrix. Our ability to understand Sjögren's syndrome and develop viable therapeutic options will depend on defining these events of epithelial cell biology.

The caveolin-1 connection to cell death and survival.

Caveolins are a family of membrane proteins required for the formation of small plasma membrane invaginations called caveolae that are implicated in cellular trafficking processes. In addition to this structural role, these scaffolding proteins modulate numerous intracellular signaling pathways; often via direct interaction with specific binding partners. Caveolin-1 is particularly well-studied in this respect and has been attributed a large variety of functions. Thus, Caveolin-1 also represents the best-characterized isoform of this family with respect to its participation in cancer. Rather strikingly, available evidence indicates that Caveolin-1 belongs to a select group of proteins that function, depending on the cellular settings, both as tumor suppressor and promoter of cellular traits commonly associated with enhanced malignant behavior, such as metastasis and multi-drug resistance. The mechanisms underlying such ambiguity in Caveolin-1 function constitute an area of great interest. Here, we will focus on discussing how Caveolin-1 modulates cell death and survival pathways and how this may contribute to a better understanding of the ambiguous role this protein plays in cancer.

Decreased phosphorylation of Y¹4caveolin-1 in endometrial tissue of polycystic ovary syndrome patients may be related with an insulin resistant state in this tissue.

Endometrial tissue of patients with polycystic ovary syndrome (PCOS) shows an impaired expression of insulin signaling molecules. Tyrosine phosphorylation of the insulin receptor (IR) by insulin promotes glucose uptake by activating the PI3K/Akt pathway. IR stability and function depend on the presence of the protein caveolin-1. Activation of IR increases phosphorylation of Y¹4caveolin-1. Since the endometrium of PCOS patients is proposed to be insulin resistant, we evaluated the phosphorylation of IR and caveolin-1 in endometria of patients with insulin resistance (PCOSE-IR) compared to controls (CE). To explore the mechanism associated with this condition, cultured endometrial cells (T-HESC) were exposed to high glucose (25 mM, 24 h), an experimental condition that leads to insulin resistance in other cell types. Endometrial protein levels of phospho-Y97²IR, phospho-Y¹4caveolin-1 and caveolin-1 were determined by Western blotting. In cultured cells, protein levels of caveolin-1, IR, and Akt were evaluated by Western blotting. After acute insulin stimulation, phospho-S47³Akt, phospho-Y¹4caveolin-1, and 2-deoxyglucose (2-DOG) uptake were determined. PCOSE-IR samples showed high protein levels of caveolin-1, but reduced phospho-Y¹4caveolin-1 compared to CE. No differences were observed for phospho-Y97²IR between both groups. Cells pretreated with glucose showed a reduction in protein levels of IR and caveolin-1 and were unable to increase 2-DOG uptake, phospho-S47³Akt and phospho-Y¹4caveolin-1 after insulin stimulation. In conclusion, in PCOSE-IR the impaired phosphorylation of IR downstream molecules such as phospho-Y¹4caveolin-1 suggests a diminished insulin sensitivity in endometria, condition that could be supported in vitro by the ability of T-HESCs to become insulin resistant when they are exposed to high glucose.

Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome.

OBJECTIVES: MUC5B contains sulfated and sialylated oligosaccharides that sequester water required for moisturising the oral mucosa. Xerostomia, in patients with Sjögren syndrome, is generally associated with reduced quantities, rather than altered properties, of saliva. Here, we determined the amount of MUC5B (mRNA and protein) as well as sulfation levels in salivary glands of patients with normal or altered unstimulated salivary flow. Localisation of MUC5B and sulfated MUC5B, as well as total levels sulfated groups were determined and compared with acini basal lamina disorganisation. PATIENTS AND METHODS: In all, 18 patients with normal or altered unstimulated salivary flow and 16 controls were studied. MUC5B mRNA and protein were evaluated in salivary glands by semiquantitative RT-PCR and Western blot analysis. MUC5B sulfation was determined by Western blotting. MUC5B and sulfo-Lewis(a) antigen localisation were assessed by immunohistochemistry. The total amount of sulfated oligosaccharides was determined microdensitometrically. RESULTS: No significant differences were detected in MUC5B mRNA and protein levels between controls and patients, while sulfo-Lewis(a) antigen levels were lower in patients. The number of sulfo-Lewis(a) positive mucous acini was reduced in patients but no correlation was observed between lower levels of sulfation and unstimulated salivary flow. Microdensitometric data confirmed the presence of reduced sulfated oligosaccharides levels in mucous acini from patients with highly disorganised basal lamina. CONCLUSION: Disorganisation of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia in these patients.

Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription.

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment beta-catenin-T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of beta-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP-CK2alpha in HEK-293T cells resulted in beta-catenin-Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented beta-catenin-Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP-CK2alpha expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP-survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2alpha in HEK-293T cells coincided with reduced beta-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via beta-catenin-Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity.

Caveolin-1 expression and function were investigated in human colon cancer. Low levels of caveolin-1 mRNA and protein were detected in several colon carcinoma cell lines. Moreover, caveolin-1 protein levels were significantly reduced in human tumor epithelial mucosa (3.6 +/- 1.4-fold) when compared with normal colon mucosa for a majority (10 of 15) of the patients characterized. To directly assess the role of caveolin-1 in tumor development, caveolin-1 was reexpressed in the HT29 and DLD1 colon carcinoma cells, and the resulting HT29-cav-1 or DLD1-cav-1 cells were tested for tumorigenicity in nude mice. In most experiments, tumor formation was either blocked or retarded for HT29-cav-1 cells (10 of 13 mice) and DLD1-cav-1 cells (5 of 7 mice), as compared with both mock-transfected and parental HT29 or DLD1 cells. Interestingly, basal caveolin-1 levels were significantly reduced in HT29-cav-1 and DLD1-cav-1 cells isolated from tumors. Likewise, endogenous caveolin-1 mRNA and protein levels were found to be reduced in NIH-3T3 cells recovered from tumors after injection into nude mice. Thus, reexpression of caveolin-1 in colon carcinoma lines reduced the probability of tumor formation in vivo, and when tumors did develop from either HT29-cav-1, DLD1-cav-1, or NIH-3T3 cells, lower basal levels of caveolin-1 were detected. Finally, evidence was obtained indicating that initial caveolin-1 down-regulation in colon cancer cells need not be an entirely irreversible process because cell survival on selection for either drug resistance or increased metastatic potential correlated with increased caveolin-1 expression levels.

Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT.

Clustering of the glycosyl-phosphatidylinositol (GPI)-anchored protein Thy-1 on the cell surface leads to T cell activation. However, despite the similarity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinking, reportedly occurs in a manner independent of the TCR/CD3 complex. To investigate the relationship between responses resulting from Thy-1 or TCR engagement, a biochemically well defined system employing only affinity purified antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium influx and IL-2 production. By these criteria, anti-CD3 mAbs moderately activated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss mutant cell line, did not respond to stimulation through CD3 despite expressing all essential signaling molecules. Together these results emphasized the existence of a poorly appreciated mutual interdependence between Thy-1 and CD3 for efficient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These biochemical properties are identical to those described for a recently cloned adaptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyclonal Abs raised against a LAT-peptide (amino acids 103-131) specifically recognized the 40 kDa protein. LAT is present in microdomains of the plasma membrane enriched in sphingolipids, cholesterol, GPI-anchored proteins and a variety of signaling molecules. By contrast, the TCR/CD3 complex is excluded from these domains at least until stimulation takes place. Hence, we propose that Thy-1 promotes TCR/CD3 dependent signaling by facilitating LAT phosphorylation on tyrosine and the subsequent recruitment of downstream effector molecules.

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

A factor with a zinc- and phorbol ester-binding domain is necessary for endosome fusion.

An inhibitory effect of several zinc chelators on endosome fusion reconstituted in an in vitro system has been recently reported (A. Aballay et al., 1995, Biochem. J. 312, 919-923). The factor that requires zinc for its activity is still unknown. Since the regulatory domain of protein kinase C (PKC) contains cysteine-rich motifs which coordinate zinc, we suspected that PKC or a PKC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a specific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13-acetate), an activator of several PKC isoforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc-regulated manner, whereas PMA enhanced endosome fusion. Moreover, fusion was strongly stimulated when both PMA and zinc were added together to zinc-depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a glutathione S-transferase fusion protein containing a cysteine-rich region of the regulatory domain of PKCgamma inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presence of proteins containing PKC-like cysteine-rich regions that are released from endosomal fractions by zinc chelators. These results indicate that the previously proposed zinc-dependent factor required for endosome fusion could be either a PKC isoform or a protein containing the phorbol ester-binding domain of PKC.

Cysteine-rich regions of protein kinase Cdelta are functionally non-equivalent. Differences between cysteine-rich regions of non-calcium-dependent protein kinase Cdelta and calcium-dependent protein kinase Cgamma.

Regulatory domain elements of the non-calcium-dependent protein kinase Cdelta (nPKCdelta), including either or both of the cysteine-rich regions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, in mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu with high affinity (Kd: 14-37 nM) and to significant stoichiometric levels (0.23-0.66 mol/mol), but no binding could be detected for proteins with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(delta) was highly cooperative with Hill numbers lying in the range of 2.5-5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCdelta and the calcium-dependent isoform, cPKCgamma, where both cysteine-rich regions represent functional PDBu binding elements.