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Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase C by diverting the kinase from a degradative pathway to the recycling endocytic compartment

Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel

View larger version (46K): [in a new window]   Fig. 1. TPA-induced downregulation of PKC. Control (empty-vector-transfected) RBL cells (A,B), RBL-Syt II- cells (A) and RBL-Syt III- cells (B) were incubated with or without TPA (50 nM) for 6 hours. Whole lysates (40 µg protein) were resolved by SDS-PAGE and immunoblotted. The cellular level of PKC was determined by western blot analysis using anti PKC antibodies. The level of Syt II was determined using the monoclonal antibody 8G2B directed against the N-terminus of Syt II. The cellular level of Syt III was determined using a rabbit polyclonal serum directed against the cytoplasmic domain of Syt III. The cellular level of actin was determined to judge for equal loading. Data represent one of three separate experiments.

View larger version (15K): [in a new window]   Fig. 2. Kinetics of TPA-induced PKC degradation. Control empty-vector-transfected RBL cells, RBL-Syt II- and RBL-Syt II+ cells were treated with TPA (50 nM) for the indicated time periods, and the cellular level of PKC was determined as described in Fig. 1. The intensities of the bands corresponding to PKC were quantified by densitometry and are presented as a percentage of the amount of enzyme present in untreated cells. Data represent the average±s.e. of three separate experiments.

View larger version (52K): [in a new window]   Fig. 3. (A) TPA-induced downregulation of PKC is sensitive to a proteasome inhibitor. Control (empty-vector-transfected) RBL cells and RBL-Syt II- cells were treated for 12 hours with TPA (50 nM) in the absence or presence of the lysosomal inhibitor (leupeptin, 10 µg/ml) or the proteasomal inhibitor (ALLN, 38.5 µg/ml). Cytosolic and membranal fractions were separated as described in the Materials and Methods, and the amount of PKC in each fraction was determined as described in Fig. 1. Data represent one of two separate experiments. (B) TPA-induced downregulation of PKC is sensitive to the PKC inhibitor Go 6976. Control (empty vector transfected) RBL cells and RBL-Syt II- cells were treated for 12 hours with TPA (50 nM) in the absence or presence of the PKC inhibitor Go 6976 (100 nM). The cellular level of PKC was determined as described in Fig. 1. Data represent one of three separate experiments.

View larger version (26K): [in a new window]   Fig. 4. Modulation of exocytosis. Control (empty-vector-transfected) RBL cells (1-4), RBL-Syt II- cells (5-8) and RBL-Syt III- cells (9-12) were incubated for 30 minutes at 37°C in the presence of buffer (1,5,9), the Ca2+ ionophore (A23187, 10 µM, 2,6,10) and the combination Ca2+ ionophore (A23187, 10 µM) and TPA (50 nM) without (3,7,11) or with (4,8,12) prior treatment with TPA (50 nM) for 6 hours. The extent of release is presented as a percentage of total cellular ß-hexosaminidase activity. Data represent the average±s.e. of three separate experiments.

View larger version (37K): [in a new window]   Fig. 5. TPA-stimulated traffic of PKC in control RBL and RBL-Syt II- cells. Upper panel: control (empty-vector-transfected) RBL cells (A) and RBL-Syt II- (B) cells were treated with TPA (50 nM) for the indicated time periods. Cell homogenates were prepared, and equal amounts of protein fractionated on continuous sucrose gradients as described in the Materials and Methods. Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-PKC antibodies. The intensities of the bands corresponding to PKC were quantified by densitometry and are presented as a percentage of the total amount of enzyme (C). Data represent one of two separate experiments.

View larger version (56K): [in a new window]   Fig. 6. Immunofluorescence analyses of TPA-dependent traffic of PKC. Control (empty-vector-transfected) RBL cells (A-J) and RBL-Syt II- cells (A'-J') were treated with TPA for the indicated time periods: 0 minutes (A,A'); 20 minutes (B-D,B'-D'); 2 hours (E-G,E'-G'); or 4 hours (H-J,H'-J'). During the last 5 minutes of incubation with TPA, FITC-Tfn (50 µg/ml) was added. The cells were subsequently processed for immunofluorescent staining using rabbit polyclonal anti-PKC antibodies (1:200 dilution) and Rhodamine-conjugated donkey anti-rabbit antibodies. The cells were visualized by confocal microscopy, as described in the Materials and Methods. Red, PKC; green, Tfn; yellow, overlap. Data represent one of six separate experiments.

View larger version (115K): [in a new window]   Fig. 7. (A) Tfn-positive endosomes co-stain with Rab 11. RBL-Syt II- cells were treated at 37°C with TPA for 2 hours and incubated with FITC-Tfn (50 µg/ml) for the last 5 minutes of incubation. The cells were subsequently processed for immunofluorescent staining using rabbit polyclonal anti Rab 11 antibodies followed by Rhodamine-conjugated donkey anti-rabbit antibodies. A-Tfn (green), B-Rab 11 (red), C-Merge. Data represent one of two separate experiments. (B) PKC-positive vesicles co-stain with serotonin. RBL (A-C) and RBL-Syt II- (D-F) cells were treated at 37°C with TPA for 4 hours. The cells were subsequently processed for immunofluorescent staining using rabbit polyclonal anti-PKC and mouse monoclonal anti-serotonin antibodies followed by Rhodamine-conjugated donkey anti-rabbit antibodies and FITC-conjugated donkey anti-mouse antibodies. A, D-PKC (red), B, E-serotonin (green), C, F-Merge. Data represent one of three separate experiments.

View larger version (21K): [in a new window]   Fig. 8. A model illustrating TPA-induced trafficking of PKC in RBL cells (A) and RBL-Syt II- cells (B). According to this model, exposure to TPA of the control cells results in translocation of PKC from the cytosol to the plasma membrane, from where the enzyme is delivered to early endosomes and subsequently a fraction is delivered to the SG, whereas most of the kinase is delivered to late endosomes and degradation. In the RBL-Syt II- cells, the transport to the late endosome and the degradative compartment is retarded because of the reduced amount of Syt II. As a consequence, the kinase is delivered to the recycling endosomal compartment, from where it recycles back to the plasma membrane. EE, early endosomes; RE, recycling endosomes, LE, late endosomes; SG, secretory granules; N, nucleus.