B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.     

Fine structure of the cell surface and Golgi apparatus of Ochromonas

Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340-360 nm long, and small projections, 50-110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthernium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.     

Dehydroascorbate and glucose are taken up into Arabidopsis thaliana cell cultures by two distinct mechanisms

The possible involvement of glucose (Glc) carriers in the uptake of vitamin C in plant cells is still a matter of debate. For the first time, it was shown here that plant cells exclusively take up the oxidised dehydroascorbate (DHA) form. DHA uptake is not affected by 6-bromo-6-deoxy-ascorbate, an ascorbate (ASC) analogue, specifically demonstrating ASC uptake in animal cells. There is no competition between Glc and DHA uptake. Moreover, DHA and Glc carriers respond in the opposite manner to different inhibitors (cytochalasin B, phloretin and genistein). In conclusion, the plant plasma membrane DHA carrier is distinct from the plant Glc transporters.     

Endocytic membrane traffic to the Golgi apparatus in a regulated secretory cell line

We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.     

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.     

Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques

High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein gylcosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.     

Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2alpha and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1-2 h and is due to phosphorylation of eIF2alpha. Continued hypoxic exposure activates a second, eIF2alpha-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT-PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2alpha. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.     

AKAP350 at the Golgi apparatus. I. Identification of a distinct Golgi apparatus targeting motif in AKAP350

The protein kinase A-anchoring proteins (AKAPs) are defined by their ability to scaffold protein kinase A to specific subcellular compartments. Each of the AKAP family members utilizes unique targeting domains specific for a particular subcellular compartment. AKAP350 is a multiply spliced AKAP family member localized to the centrosome and the Golgi apparatus. Three splicing events in the carboxyl terminus of AKAP350 generate the AKAP350A, AKAP350B, and AKAP350C proteins. A monoclonal antibody recognizing all three splice variants as well as a polyclonal antibody specific for AKAP350A demonstrated both centrosomal and Golgi apparatus staining in paraformaldehyde-fixed HCA-7 cells. Golgi apparatus-associated AKAP350A staining was dispersed following brefeldin A treatment. Using GFP chimeric constructs of the carboxyl-terminal regions of AKAP350A, a Golgi apparatus targeting domain was identified between amino acids 3259 and 3307 of AKAP350A. This domain was functionally distinguishable from the recently described centrosomal targeting domain (PACT domain, amino acids 3308-3324) located adjacent to the Golgi targeting domain. These data definitively establish the specific association of AKAP350A with the Golgi apparatus in HCA-7 cells.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

The Golgi protein RCAS1 controls cell surface expression of tumor-associated O-linked glycan antigens

Tumor immunology has received a large impetus from the identification of tumor-associated antigens. Among them, a monoclonal antibody, 22.1.1, was instrumental in defining a novel tumor-associated antigen that was termed "receptor binding cancer antigen expressed on SiSo cells" (RCAS1). RCAS1 was proposed to induce growth arrest and apoptosis on activated immune cells, mediated by a putative death receptor. Structurally, RCAS1 was predicted to exist as a type II transmembrane protein and in a soluble form. Here, we analyzed occurrence, membrane topology, and subcellular localization of the RCAS1-encoded gene product. RCAS1 was shown to be a ubiquitously expressed type III transmembrane protein with a Golgi-predominant localization. Monoclonal antibody 22.1.1 failed to recognize RCAS1, as demonstrated by confocal microscopy. Instead, we showed that the cognate 22.1.1 epitope is identical with the tumor-associated O-linked glycan Tn (N-acetyl-d-galactosamine, GalNAc). Overexpression of RCAS1 in cell lines that are negative for 22.1.1 surface staining led to the generation of Tn and the closely related TF (Thomsen-Friedenreich, Galbeta1-3GalNAc) antigen, thus providing a functional link to the generation of the 22.1.1 epitope. We suggest that RCAS1 modulates surface expression of tumor-associated, normally cryptic O-linked glycan structures and contributes indirectly to the antigenicity of tumor cells.     

TNF receptor 1 mediates dendritic cell maturation and CD8 T cell response through two distinct mechanisms

TNF-α and its two receptors (TNFR1 and 2) are known to stimulate dendritic cell (DC) maturation and T cell response. However, the specific receptor and mechanisms involved in vivo are still controversial. In this study, we show that in response to an attenuated mouse hepatitis virus infection, DCs fail to mobilize and up-regulate CD40, CD80, CD86, and MHC class I in TNFR1(-/-) mice as compared with the wild-type and TNFR2(-/-) mice. Correspondingly, virus-specific CD8 T cell response was dramatically diminished in TNFR1(-/-) mice. Adoptive transfer of TNFR1-expressing DCs into TNFR1(-/-) mice rescues CD8 T cell response. Interestingly, adoptive transfer of TNFR1-expressing naive T cells also restores DC mobilization and maturation and endogenous CD8 T cell response. These results show that TNFR1, not TNFR2, mediates TNF-α stimulation of DC maturation and T cell response to mouse hepatitis virus in vivo. They also suggest two mechanisms by which TNFR1 mediates TNF-α-driven DC maturation, as follows: a direct effect through TNFR1 expressed on immature DCs and an indirect effect through TNFR1 expressed on naive T cells.     

Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms

The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.