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• W2166719652 endingPage "1997" @default.
• W2166719652 startingPage "1991" @default.
• W2166719652 abstract "Studies of RIIα-deficient B lymphoid cells and stable transfectants expressing the type IIα regulatory subunit (RIIα) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type IIα mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RIIα-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RIIα in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type IIα seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RIIα expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RIIα were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type IIα isozyme. Studies of RIIα-deficient B lymphoid cells and stable transfectants expressing the type IIα regulatory subunit (RIIα) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type IIα mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RIIα-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RIIα in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type IIα seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RIIα expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RIIα were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type IIα isozyme. protein kinase A trans Golgi network endoplasmic reticulum recombinant ricin with a tyrosine sulfation site recombinant ricin construct with a sulfation site and three overlappingN-glycosylation sites fluorescein isothiocyanate fetal calf serum 8-(4-chlorophenylthio)-cAMP 2-mercaptoethanesulfonic acid monoclonal antibody phosphate-buffered saline The mechanism by which different extracellular ligands that mediate their signals through the same second messenger might give rise to a specific intracellular response has been the subject of intensive research for several years (1Skalhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Crossref PubMed Google Scholar). In the case of cAMP signal transduction, it has been demonstrated that the subcellular localization of protein kinase A (PKA)1 is important for the specificity (1Skalhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Crossref PubMed Google Scholar). PKA is composed of two catalytic (C) subunits and one regulatory dimer (R2) that in the absence of cAMP form an inactive heterotetramer (R2C2). Upon binding of cAMP to the R subunits, the enzyme dissociates and releases two free, active C subunits (2Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (411) Google Scholar). The R2 dimer is also implicated in the targeting of different PKA isoforms to various intracellular locations and to specific substrates through interactions with protein kinase A-anchoring proteins (3Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Four different isoforms of the R subunit have been identified as products of separate genes in mammalian cells, and they have been termed RIα, RIβ, RIIα, and RIIβ (1Skalhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Crossref PubMed Google Scholar, 2Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (411) Google Scholar, 4Oyen O. Myklebust F. Scott J.D. Hansson V. Jahnsen T. FEBS Lett. 1989; 246: 57-64Crossref PubMed Scopus (56) Google Scholar). They all contain two C-terminal cAMP binding sites, a hinge region that interacts with and inhibits the catalytic subunit, and a dimerization domain responsible for the interaction between the two regulatory subunits that make up the regulatory dimer of PKA (2Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (411) Google Scholar). Whereas RI subunits are known to be mainly soluble, RII subunits are primarily associated with cytoskeletal elements and membranes (1Skalhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Crossref PubMed Google Scholar, 3Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Studies of several human cell lines have revealed that the PKA type IIα isozyme (containing RIIα and Cα) is concentrated in centrosomes and in Golgi-associated compartments (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar, 6Martin M.E. Hidalgo J. Vega F.M. Velasco A. J. Cell Sci. 1999; 112: 3869-3878PubMed Google Scholar). In contrast, PKA type IIβ (containing RIIβ) is associated more selectively with the centrosomal region and not with Golgi structures (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar). Based on the important role of the Golgi apparatus in intracellular transport and protein sorting and the localization of PKA, which previously has been implicated in vesicle-mediated protein transport processes (7Pimplikar S.W. Simons K. J. Biol. Chem. 1994; 269: 19054-19059Abstract Full Text PDF PubMed Google Scholar, 8Hansen S.H. Casanova J.E. J. Cell Biol. 1994; 126: 677-687Crossref PubMed Scopus (118) Google Scholar, 9Mostov K.E. Cardone M.H. Bioessays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar, 10Eker P. Holm P.K. van Deurs B. Sandvig K. J. Biol. Chem. 1994; 269: 18607-18615Abstract Full Text PDF PubMed Google Scholar), the possibility existed that a distinct Golgi-associated pool of PKA type IIα isozyme was involved in regulation of transport through this organelle. To investigate whether PKA type IIα is involved in the regulation of retrograde transport, we studied the transport of the plant toxin ricin. Ricin belongs to a family of plant and bacterial toxins that enter cells via the endocytic pathway. The toxin is transported retrogradely through the Golgi to the endoplasmic reticulum (ER) before it enters the cytosol, where it inhibits protein synthesis (11Sandvig K. van Deurs B. Physiol. Rev. 1996; 76: 949-966Crossref PubMed Scopus (266) Google Scholar, 12Sandvig K. van Deurs B. EMBO J. 2000; 19: 5943-5950Crossref PubMed Google Scholar). Ricin consists of an A-chain and a B-chain that are linked by a disulfide bridge. The B-chain binds to terminal galactose in both glycolipids and glycoproteins at the plasma membrane, whereas the A-chain enzymatically inhibits the protein synthesis after entry into the cytosol (12Sandvig K. van Deurs B. EMBO J. 2000; 19: 5943-5950Crossref PubMed Google Scholar). Because ricin binds to both glycolipids and glycoproteins at the plasma membrane, it will be endocytosed by any vesicle that pinches off. Once ricin is endocytosed, it can be transported through the endosomal compartments, recycled back to the plasma membrane, delivered to the lysosomes, or transported retrogradely to the TGN and to the ER (11Sandvig K. van Deurs B. Physiol. Rev. 1996; 76: 949-966Crossref PubMed Scopus (266) Google Scholar,12Sandvig K. van Deurs B. EMBO J. 2000; 19: 5943-5950Crossref PubMed Google Scholar). In this study, we took advantage of a RIIα-deficient B lymphoid cell line, Reh (13Tasken K. Skalhegg B.S. Solberg R. Andersson K.B. Taylor S.S. Lea T. Blomhoff H.K. Jahnsen T. Hansson V. J. Biol. Chem. 1993; 268: 21276-21283Abstract Full Text PDF PubMed Google Scholar), and reintroduced RIIα by making stable transfectants with a Golgi-associated pool of PKA type IIα to examine the effect on intracellular transport. We used two different ricin constructs to monitor the retrograde toxin transport. Within the Golgi apparatus, recombinant ricin with a tyrosine sulfation site (ricin sulf-1) becomes radiolabeled in the presence of radioactive sulfate (14Rapak A. Falnes P.O. Olsnes S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3783-3788Crossref PubMed Scopus (219) Google Scholar). This has made ricin sulf-1 a valuable tool to study intracellular transport to the TGN. A recombinant ricin construct with a sulfation site and three overlapping N-glycosylation sites (ricin sulf-2) is both modified in the TGN and N-glycosylated in the ER (14Rapak A. Falnes P.O. Olsnes S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3783-3788Crossref PubMed Scopus (219) Google Scholar). TheN-glycosylation of the toxin results in a molecular shift that can be observed on SDS-PAGE. This construct has therefore been used to study intracellular transport to the ER. As shown in the present study, the expression of PKA type IIα (RIIα) on a negative background in a lymphoid cell line leads to modulation of the retrograde transport of ricin, indicating a regulatory role for Golgi-associated PKA type IIα on these transport steps. [3H]Leucine and Na235SO4 were purchased fromAmersham Biosciences. Na125I was purchased from DuPont. Hygromycin B was bought from Roche Molecular Biochemicals (Mannheim, Germany) and Mowiol was obtained from Calbiochem. Cy3-labeled goat anti-rabbit, FITC-labeled goat anti-mouse, and FITC-labeled goat anti-rabbit were purchased from Jackson ImmunoResearch (West Grove, PA). Fetal calf serum (FCS), RPMI 1640 medium, RPMI 1640 medium without sulfate, and streptomycin were bought from Invitrogen and protein A-Sepharose was purchased from Pharmacia. 8-(4-Chlorophenylthio)-cAMP (8CPT-cAMP), aprotinin, cycloheximide, HEPES, lactose, MESNA, phenylmethylsulfonyl fluoride, poly(d-lysine) (M r = 150,000), ricin, and ricin B-chain were obtained from Sigma Chemical. Rabbit anti-human RIIα and RIIβ polyclonal antibodies and mouse anti-RIIα monoclonal antibodies have been described elsewhere (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar). Medial-Golgi (mAb CTR 433) and centrosomal (mAb CTR 453) markers were kindly provided by Dr. Michel Bornens (Institute Curie, Paris, France). The cis Golgi marker GM130 was obtained from BD Biosciences. The trans Golgi marker TGN46 was obtained from Serotec (Oxford, UK). A human B-lymphoid cell line (Reh) stably transfected with pMep4 vector (clone pMep) or RIIα under direction of the human metallothionein IIA promoter (clone RIIα) (15Carlson C.R. Witczak O. Vossebein L. Labbé J.-C. Skålhegg B.S. Keryer G. Herberg F.W. Collas P. Tasken K. J. Cell Sci. 2001; 114: 3243-3254PubMed Google Scholar) was maintained under standard conditions (5% CO2 in RPMI 1640 medium containing 5% (v/v) FCS, 2 mm l-glutamine, and 100 μg/ml streptomycin). Every third month, the cells expressing RIIα were incubated under standard conditions in the presence of 200 μg/ml hygromycin B. On the day that the experiments were performed, the cells were seeded into Eppendorf tubes at a density of 8 × 105 cells/tube. The cells were washed twice with HEPES medium (bicarbonate-free Eagle's minimum essential medium buffered with 20 mm HEPES to pH 7.4), and then incubated with the same medium for 30 min at 37 °C. The samples were then incubated in the presence or absence of 350 μm8CPT-cAMP for 30 min before 1, 10, and 100 ng/ml of ricin were added to the cells, which were further incubated for 30 min at 37 °C. The cells were incubated thereafter with HEPES medium containing 1 μCi/ml [3H]leucine for 20 min at 37 °C, extracted with 5% (w/v) trichloroacetic acid for 10 min followed by a brief wash with the same solution. Subsequently, cells were dissolved in 0.1m KOH, and the acid-precipitable radioactivity was measured. The results are presented as percentage of radioactivity incorporated in cells incubated without toxin. The concentration of ricin required to inhibit the protein synthesis by 50% was chosen as a measure of the sensitivity of cells to ricin. Variation between duplicate measurements was less than 15%. Recombinant ricin A-sulf-1 and ricin A-sulf-2, modified to contain a tyrosine sulfation site and both a tyrosine sulfation site and three overlappingN-glycosylation sites, respectively, were expressed, purified, and reconstituted with ricin B-chain (ricin sulf-1 and ricin sulf-2, respectively) according to the procedure described previously (14Rapak A. Falnes P.O. Olsnes S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3783-3788Crossref PubMed Scopus (219) Google Scholar). The cells were washed twice in sulfate-free RPMI 1640 medium that contained 2 mm l-glutamine, and then incubated with 0.1 mCi/ml Na235SO4 in the same medium for 3 h. The cells were then incubated in the presence or absence of 350 μm 8CPT-cAMP and/or 20 μg/ml cycloheximide for 30 min at 37 °C, before ricin sulf-1 or ricin sulf-2 (∼300 ng/ml) was added. The incubation was continued for 2 h at 37 °C. The cells were then washed twice for 5 min at 37 °C with HEPES medium that contained 0.1 m lactose followed by cold PBS (140 mm NaCl and 10 mmNa2HPO4, pH 7.2). The cells were thereafter lysed (lysis buffer, 0.1 m NaCl, 10 mmNa2HPO4, 1 mm EDTA, 1% (v/v) Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 1 mm aprotinin, pH 7.4), and centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was immunoprecipitated overnight at 4 °C with rabbit anti-ricin antibodies immobilized on protein A-Sepharose. The beads were then washed twice with PBS containing 0.35% (v/v) Triton X-100, and the immunoprecipitated material was analyzed by SDS-PAGE (12%) under reducing conditions. SDS-PAGE was carried out in the presence of β-mercaptoethanol as described previously (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar). The gels were fixed in 4% acetic acid (v/v) and 27% (v/v) methanol for 30 min and then incubated with 1 m sodium salicylate, pH 5.8, in 2% (v/v) glycerol for 30 min. The dried gels were then exposed to Kodak XAR-5 films (Eastman Kodak Co.) at −80 °C for autoradiography. For analysis of ricin distribution, ricin was labeled with Cy5 (Amersham Biosciences) according to the manufacturer's instructions. The coverslips were coated with poly(d-lysine) (M r = 150 000) as described previously (17Huang W.M. Gibson S.J. Facer P., Gu, J. Polak J.M. Histochemistry. 1983; 77: 275-279Crossref PubMed Scopus (291) Google Scholar). The cells were washed twice with HEPES medium before addition of Cy5-labeled ricin (∼1000 ng/ml). After incubating the cells for 30 min at 37 °C, they were washed with cold PBS and further incubated with 3% (w/v) paraformaldehyde in PBS for 15 min at room temperature. The cells were then washed 3 times with PBS before incubation with 0.1% (v/v) Triton X-100 dissolved in PBS for 5 min at room temperature. Subsequently, the cells were washed in PBS, and incubated with PBS containing 5% (v/v) FCS for 30 min. The permeabilized cells were incubated with rabbit anti-human RIIα (1:1000) or with mouse mAb CTR 433 (1:10) to label the medial Golgi compartment, with mouse mAb GM130 (1:1000) to label the cis Golgi, or with sheep anti-human TGN46 (1:100) to label the trans Golgi in PBS containing 5% (v/v) FCS for 30 min at room temperature. The cells were then washed three times for 5 min with PBS containing 5% (v/v) FCS followed by incubation with FITC-labeled goat anti-mouse antibody (1:100) to detect CTR 433 and GM130, with Cy3-labeled goat anti-rabbit antibody (1:500) to detect RIIα, or with FITC-labeled donkey anti-sheep/goat antibody (1:100) to detect TGN46 in PBS containing 5% (v/v) FCS. After staining, the cells were washed three times for 5 min with PBS at room temperature, and the coverslips were mounted in Mowiol. Immunofluorescence microscopy was performed using a Leica (Wetzlar, Germany) confocal microscope. Images were captured with a resolution of 1024 × 768 pixels and prepared with the use of Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA). For analysis of PKA distribution versus centrosomal marker, cells were fixed in 4% paraformaldehyde in PBS for 20 min at 37 °C, rinsed twice in PBS, and incubated for 10 min with 50 mmammonium chloride in PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS with 0.2% BSA. Primary antibodies were diluted in PBS containing 3% BSA to concentrations of 140 ng/ml for mAb CTR 453 (a centrosomal marker), 100 ng/ml for rabbit anti-human RIIα, 500 ng/ml for rabbit anti-human RIIβ, and 1 μg/ml for mouse anti-RIIα mAb and incubated for 1 h at room temperature. Cells were then washed three times in PBST (PBS with 1% Tween 20) to remove unbound antibodies followed by incubation with fluorochrome-conjugated secondary antibodies (FITC and Texas Red) for 1 h at room temperature. Finally, the cells were mounted in CITIFLUOR (Citifluor, London, UK). Confocal microscopy was performed on a Sarastro 2000 confocal microscope (Amersham Biosciences), equipped with an argon laser (488 to 514 nm wavelength). Ten sections of 0.25 μm (averaging five full frames of the same section) were scanned, and stacks of optical sections for each data set were compiled with Voxel View software on an IRIS 4D-70 GT graphics work station (SGI, Mountain View, CA). Ricin was125I-labeled according to the procedure described by Fraker and Speck (18Fraker P.J. Speck J.C., Jr. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar) to a specific activity of 5 × 104cpm/ng. The intracellular accumulation of 125I-labeled ricin was measured after 2 h at 37 °C as the amount of toxin that could not be removed by lactose treatment, as described previously (19Sandvig K. Olsnes S. Exp. Cell Res. 1979; 121: 15-25Crossref PubMed Scopus (82) Google Scholar). The cells were preincubated with 350 μm 8CPT-cAMP for 30 min at 37 °C. The endocytosis of ricin was measured using the ORIGEN analyzer (IGEN Inc., Rockville, MD). Ricin was labeled with N-hydroxysuccinimide ester-activated tris(bipyridine) chelated ruthenium(II) TAG (IGEN Inc.) according to the manufacturer's instructions and simultaneously biotinylated with reducible immunopure NHS-SS-Biotin (Pierce). The cells were washed with HEPES medium and then incubated in the presence or absence of 350 μm 8CPT-cAMP for 30 min at 37 °C, followed by addition of TAG-labeled ricin (25 ng/ml) to allow endocytic uptake of the toxin for 30 min at 37 °C. Half of the samples were then treated with 0.1m MESNA for 1 h on ice (20Smythe E. Redelmeier T.E. Schmid S.L. Methods Enzymol. 1992; 219: 223-234Crossref PubMed Scopus (40) Google Scholar), and the other half of the samples was washed in cold PBS. The cells were then lysed (lysis buffer, 100 mm NaCl, 5 mm MgCl2, 50 mm HEPES, and 1% (v/v) Triton X-100) for 10 min on ice. Ricin that is both TAG-labeled and biotinylated can be detected in the lysate by using streptavidin-conjugated beads (Dynal, Oslo, Norway) and the ORIGEN analyzer (IGEN Inc.). The endocytosis of TAG-labeled ricin was measured as the amount of toxin that could not be removed by MESNA treatment as described previously (21Skretting G. Torgersen M.L. van Deurs B. Sandvig K. J. Cell Sci. 1999; 112: 3899-3909Crossref PubMed Google Scholar). Detection of PKA subunits by Northern blot analysis or [32P]8-azido-cAMP photoaffinity labeling and immunoprecipitation was performed essentially as described previously (13Tasken K. Skalhegg B.S. Solberg R. Andersson K.B. Taylor S.S. Lea T. Blomhoff H.K. Jahnsen T. Hansson V. J. Biol. Chem. 1993; 268: 21276-21283Abstract Full Text PDF PubMed Google Scholar). To study effects related specifically to expression of RIIα, stably transfected cell lines expressing RIIα under control of the zinc-inducible type IIa metallothionein promoter were made together with control clones transfected with empty plasmid by selection on hygromycin B. Fig. 1 shows characterization of one clone expressing RIIα compared with a control clone. The RIIα transfected clone displayed high levels of RIIα mRNA (A) as well as a cAMP-binding protein, immunoreactive with RIIα antibodies and present in both the soluble S200 and detergent-soluble Tx-100 fractions (B). In contrast, a control-transfected clone did not have any detectable RIIα mRNA or protein. Both cell clones had equal levels of RIIβ mRNA. We conclude that the RIIα-transfected clone expresses RIIα at quite high levels even in the absence of zinc. It has previously been shown that RIIα is localized to the Golgi-centrosomal area in SaOS2 osteosarcoma cells and in COS-7 cells (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar, 6Martin M.E. Hidalgo J. Vega F.M. Velasco A. J. Cell Sci. 1999; 112: 3869-3878PubMed Google Scholar). We have examined, by immunofluorescence and confocal microscopy, whether this was also the case when RIIα was expressed in Reh cells. Double staining of the centrosomal marker CTR 453 (22Bailly E. Doree M. Nurse P. Bornens M. EMBO J. 1989; 8: 3985-3995Crossref PubMed Scopus (286) Google Scholar) (Figs. 2, A,C, and E) and RIIα (Figs. 2, B,D, and F) demonstrated that RIIα was absent in wild-type cells (Fig. 2 B), but appeared in the centrosomal region when expressed (Fig. 2, D and F versus C and E). Furthermore, the distribution of RIIα was wider than that of the centrosomal protein CTR 453 (Fig. 2, F versus E). Because RIIβ is targeted to centrosomes and present in Reh cells, as in most cancer cell lines [Fig. 1; Ref. 5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar), we next examined the distribution of RIIα versus that of RIIβ by dual staining and image overlays (Fig. 2 G). Again, RIIα (red) was found in a wider area than RIIβ (green, overlap seemsyellow), which in separate experiments showed a distribution overlapping well with that of the centrosomal marker, CTR 453 (not shown). In addition, we performed immunofluorescence studies of the localization of RIIα and the Golgi markers GM130 and TGN46. As shown in Fig. 3, RIIα was partially colocalized with both Golgi markers. Together, these experiments demonstrated that clones expressing RIIα acquired a new PKA isozyme that localizes in the centrosomal-Golgi region. To explore the function of this particular pool of Golgi-associated PKA, we next examined the intracellular transport of ricin in Reh cells in the presence and absence of a Golgi-associated pool of PKA type IIα.Figure 3RIIα expressed in an RIIα-deficient, B lymphoid cell line, Reh, colocalizes with Golgi markers. RIIα cells were fixed, permeabilized, double stained with hRIIα antibodies (red,A and D) and with the cis Golgi marker GM130 (green, B) or the trans Golgi marker TGN46 (green, E), and analyzed in a confocal microscope. Merged images are shown in C andF.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether one or more steps along the intracellular route followed by ricin are affected by RIIα, we tested the sensitivity to ricin of clone RIIα cells with and without the stimulation of PKA and compared it with that of cells deficient in PKA type IIα (clone pMep). To activate PKA, we used a cell-permeable cAMP analog, 8CPT-cAMP, with high affinity for the type II regulatory subunits (23Ogreid D. Ekanger R. Suva R.H. Miller J.P. Doskeland S.O. Eur. J. Biochem. 1989; 181: 19-31Crossref PubMed Scopus (122) Google Scholar, 24Ogreid D. Doskeland S.O. FEBS Lett. 1981; 129: 287-292Crossref PubMed Scopus (56) Google Scholar). As shown in Fig. 4, cells expressing RIIα were ∼2-fold more sensitive to ricin than the RIIα-deficient cells, and although addition of 8CPT-cAMP had a sensitizing effect on the pMep cells, it sensitized the cells expressing RIIα to a much larger extent. Similar experiments with untransfected cells (Reh), as well as with other clones transfected with RIIα with similar targeting, showed the same pattern of sensitivity (results not shown). Thus, RIIα regulates one or several steps on the route of ricin to the cytosol in lymphoid cells, although RIIα is not strictly required for ricin intoxication. To study the transport of ricin from the plasma membrane to the Golgi apparatus, recombinant ricin sulf-1 that contains a tyrosine sulfation site was used. The cells were incubated in the presence or absence of 8CPT-cAMP and ricin sulf-1 (Fig. 5). Because the protein synthesis was somewhat stimulated in cells preincubated with 8CPT-cAMP, and an increased transport of newly synthesized proteins through the TGN could in theory result in an increased competition for sulfation and thus interfere with the assay; some cells were also incubated with cycloheximide to inhibit protein synthesis. Fig. 5C shows that the sulfation of ricin in control cells increased by ∼70% in the presence of 8CPT-cAMP, whereas the sulfation increased by ∼120% in the cells expressing RIIα. However, in cells treated with both 8CPT-cAMP and cycloheximide, the sulfation of ricin increased by ∼80% in the control cells and by ∼250% in the RIIα-expressing cells. This result indicates that when PKA is activated, the transport of ricin to the Golgi apparatus is increased to a larger extent in cells expressing RIIα than in control cells. To further investigate the transport of ricin to the ER, recombinant ricin sulf-2 that contained a tyrosine sulfation site and three overlapping N-glycosylation sites was used. When ricin sulf-2 was added to control cells (clone pMep) or to RIIα-expressing cells (clone RIIα) in the presence of radioactive sulfate and immunoprecipitated from cell lysates, two bands were visible (Figs. 6, A andB). The upper molecular weight band represents ricin that has been both sulfated and glycosylated, and the lower molecular weight band represents ricin that has only been sulfated. As shown in Fig. 6C, the amount of ricin in the ER, measured as sulfated and glycosylated ricin relative to the total amount of sulfated ricin, was significantly increased in cells expressing RIIα (clone RIIα) compared with the control cells (clone pMep) when PKA was activated by 8CPT-cAMP. These observations indicate that not only the transport of ricin to the Golgi apparatus but also the further transport of the toxin to the ER is increased by RIIα in the presence of 8CPT-cAMP. The increased sulfation of ricin sulf-1 and ricin sulf-2 observed in cells expressing RIIα compared with control cells deficient in RIIα could be caused by an increased binding and endocytosis of ricin or by an increased endosome-to-Golgi transport. We therefore investigated the endocytosis of ricin in the presence or absence of 8CPT-cAMP. Fig. 7 demonstrates that the accumulation of ricin after 2 h of incubation was not significantly changed by addition of 8CPT-cAMP or by the expression of RIIα. Similar data were obtained when the cells were incubated with ricin for 30 min (results not shown). In addition, the binding of ricin to the plasma membrane was not significantly altered by 8CPT-cAMP or by expression of RIIα (data not shown). Thus, the increased transport of ricin to the Golgi apparatus cannot be accounted for by a change in the endocytosis of ricin. As evident from Figs. 2 and 3 and previous reports (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar), RIIα mainly exhibits a perinuclear, Golgi-associated localization that is detergent-extractable, indicating membrane-associated localization. To investigate whether RIIα is associated with ricin-containing structures, immunofluorescence studies were performed using antibodies raised against human RIIα (Fig. 8 A, visualized by Cy3-labeled secondary antibodies, red), Cy5-labeled ricin (Fig. 8 B, blue), and the medial Golgi marker CTR 433 (Fig. 8 C, visualized by FITC-labeled secondary antibodies,green). Both RIIα and ricin seemed colocalized (Fig. 8 F) and were also found to partly colocalize with the medial Golgi marker (Fig. 8, D and E, respectively). It has been reported that both the type IIα and IIβ regulatory subunits are associated with centrosomes (5Keryer G. Skalhegg B.S. Landmark B.F. Hansson V. Jahnsen T. Tasken K. Exp. Cell Res. 1999; 249: 131-146Crossref PubMed Scopus (40) Google Scholar). Because the previous experiment showed colocalization between ricin and RIIα, we investigated the distribution and localization of ricin in the centrosomal area. We showed that whereas minor amounts of RIIα are present in centrosomes, no ricin was detected in this region (data not shown). In the present study, we have investigated the influence of the Golgi-associated type IIα regulatory subunit of PKA on the intracellular transport of ricin. RIIα was expressed on a negative background, and 8CPT-cAMP was used to activate PKA. Because phenotypic differences between clones may arise (e.g. relating to incorporation in the genomic DNA), we investigated several clones expressing RIIα with similar results. Different clones transfected with empty vector displayed a phenotype similar to that of wild-type cells. The first indication that RIIα might be involved in regulation of intracellular transport was found by investigating the entry of the plant toxin ricin into the cytosol, measured as ricin toxicity. Even when PKA was not activated by addition of external 8CPT-cAMP, the cells expressing RIIα were about 2-fold more sensitive to ricin than the control cells. The most likely explanation seems to be that there is a certain level of endogenous cAMP that partly activates PKA. This explanation was strengthened by the finding that the transfected cells were about 4-fold more sensitive to ricin than the control cells when PKA was activated by addition of external 8CPT-cAMP. Also, the control cells were shown to be slightly more sensitive to ricin when PKA was activated. This might be caused by the activation of other isozymes of PKA that also regulate intracellular transport but, apparently, less efficiently than PKA type IIα. The PKA type IIα might be a better regulator than the other PKA isozymes because it is closer to the vesicular route of ricin transport into the Golgi apparatus. It might be a question of the local concentration of PKA whether it serves as a good regulator. It has previously been shown that addition of 8-bromo-cAMP to Madin-Darby canine kidney cells gives a selective stimulation of the transport of apically internalized ricin to the Golgi apparatus (25Llorente A. van Deurs B. Sandvig K. FEBS Lett. 1998; 431: 200-204Crossref PubMed Scopus (12) Google Scholar) (Fig. 9). However, in those cells, we cannot ascribe this regulation to a Golgi-located PKA. We here demonstrate that the sulfation of ricin is increased in cells expressing RIIα compared with control cells when PKA was activated by externally added 8CPT-cAMP. This result indicates that RIIα is a strong regulator of the transport of ricin from the plasma membrane to the Golgi apparatus. Earlier studies have demonstrated that calmodulin (26Llorente A. Garred Ø. Holm P.K. Eker P. Jacobsen J. van Deurs B. Sandvig K. Exp. Cell Res. 1996; 227: 298-308Crossref PubMed Scopus (30) Google Scholar) and calcium (27Lauvrak S.U. Llorente A. Iversen T.G. Sandvig K. J. Cell Sci. 2002; 115: 3449-3456PubMed Google Scholar) can modulate the transport of ricin from endosomes to the Golgi apparatus in other cell lines (Fig. 9). Clearly, different factors are involved in the regulation of retrograde transport. Interestingly, confocal microscopy demonstrated a localization of both ricin and RIIα in the Golgi area. The difference in ricin sulfation was much larger when both cell types were preincubated in the presence of cycloheximide. Such experiments were performed because the protein synthesis was somewhat stimulated in cells incubated with 8CPT-cAMP (data not shown), and an increased transport of newly synthesized proteins through the TGN in theory could result in an increased competition for sulfation. Even though it cannot be excluded that cycloheximide might result in an increased transport of ricin to the Golgi apparatus, the increased sulfation of ricin that was observed in cells expressing RIIα compared with the control cells in the presence of 8CPT-cAMP and cycloheximide strongly supports the notion of a regulatory role of RIIα in intracellular transport from the plasma membrane to the Golgi apparatus. The increased transport of ricin to the Golgi apparatus in cells expressing RIIα could be caused by increased binding and endocytosis of the toxin. However, no significant stimulation of the binding or the endocytosis of the toxin after 30 min or 2 h of incubation were observed in the RIIα-expressing cells, strongly indicating a selective regulatory role of RIIα in the transport of ricin from the endosomal compartments to the Golgi apparatus and further to the ER. At the moment, we can only speculate about the molecular mechanism of the regulation of the endosome to Golgi transport of ricin by PKA type IIα. There are several examples of the importance of phosphorylation for transport. For example, it has been shown that the activity of PKA has an effect on the in vitro association of ARF1 to Golgi membranes (28Martin M.E. Hidalgo J. Rosa J.L. Crottet P. Velasco A. J. Biol. Chem. 2000; 275: 19050-19059Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It is possible that upon stimulation, the Golgi-associated PKA type IIα phosphorylates membrane proteins in neighboring compartments that recruit cytosolic proteins involved in the trafficking of ricin between endocytic organelles and the Golgi apparatus. Another possibility is that PKA type IIα is important for the fusion of incoming vesicles with the Golgi apparatus. Transport of ricin not only to the TGN, where the sulfotransferase is located (29Leitinger B. Brown J.L. Spiess M. J. Biol. Chem. 1994; 269: 8115-8121Abstract Full Text PDF PubMed Google Scholar), but also from the Golgi apparatus to the ER, measured as ricin that has been both sulfated and glycosylated, was shown to increase in cells expressing RIIα relatively to the control cells in the presence of 8CPT-cAMP. An increased amount of glycosylated ricin could have been a result of the stimulated transport to the Golgi apparatus of this toxin. However, the fraction of ricin that has been both sulfated and glycosylated compared with the total amount of sulfated ricin is also increased in cells expressing RIIα in the presence of 8CPT-cAMP, thus indicating an additional regulatory role of RIIα in the transport of ricin from the Golgi to the ER. In conclusion, our results indicate that the Golgi-associated type IIα regulatory subunit of PKA regulates endosome-to-Golgi and Golgi-to-ER transport of ricin in lymphocytes." @default.
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• W2166719652 title "Endosome-to-Golgi Transport Is Regulated by Protein Kinase A Type IIα" @default.
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