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First published online 3 March 2009
doi: 10.1242/jcs.041061

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Mitochondrial diacylglycerol initiates protein-kinase-D1-mediated ROS signaling

¹ Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Griffin Building, Room 306, 4500 San Pablo Road, Jacksonville, FL 32224, USA
² Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
³ Department of Chemistry, School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (35K): [in this window] [in a new window]   Fig. 1. Translocation and activation of PKD1 by mitochondrial oxidative stress. (A) HeLa cells were transfected with pDsRed2-Mito and HA-tagged PKD1 and stimulated with Rotenone (20 µM, 1 hour) or left untreated (control). Localization of PKD1 and mitochondria was analyzed by confocal microscopy. (B) HeLa cells were transfected with wild-type PKD1 and stimulated with H2O2 (10 mM, 10 minutes) or Rotenone (20 µM, 1 hour). PKD1 was immunoprecipitated, samples were separated on SDS-PAGE and transferred to nitrocellulose. Samples were analyzed by western blotting with -pS738/S742 (phosphorylated activation loop). Total PKD1 was detected by stripping and re-probing for PKD1. All experiments were performed three times with similar results.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Oxidative stress induces mitochondrial DAG formation. (A,B) HeLa cells were transfected with a mitochondrial DAG reporter (Daglas-mit1). Cells were stimulated over time with 10 mM H2O2. H2O2-induced mitochondrial DAG formation was analyzed by measuring the CFP/YFP intensity ratio using a confocal microscope. (A) Real-time imaging of DAG formation at the mitochondria. Shown are CFP and YFP mitochondrial signals at start time (T0) and end time (T30=870 seconds). The decrease of CFP signal and increase of YFP signal indicates FRET caused by mitochondrial DAG formation. (B) Quantification of the experiment by depicting the CFP/YFP intensity ratio. The arrow indicates the start time for H2O2 stimulation (T6=150 seconds). All experiments were performed several times and with similar results.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Functional N-terminal and C1 domains in PKD1 are required for its activation by oxidative stress. (A) Overview PKD1 structure and mutants. PKD1 consists of two C1 domains, C1a and C1b (CRD domain), a pleckstrin homology domain (PH) and a kinase domain (KD). PKD1 deletion and point mutants were generated to examine the importance of DAG binding on PKD1 activation in response to oxidative stress. PKD1.1-321: PKD1 mutant with deletion of the first 321 amino acids of the N-terminal domain. PKD1.CRD: PKD1 mutant with deletion of the region comprising C1a and C1b (CRD domain). PKD1.P157G: PKD1 point mutant with a P to G mutation that blocks DAG binding by the C1a domain, but leaves the tertiary structure of PKD1 intact. PKD1.P281G: PKD1 point mutant with a P to G mutation that blocks DAG binding by the C1b domain. (B) HeLa cells were transfected with vector control, wild-type PKD1 or PKD1.1-321. Cells were stimulated with H2O2 (10 mM, 10 minutes) as indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blotting was performed with -pS738/S742. Total PKD1 was detected by stripping and re-probing for PKD1. (C) HeLa cells were transfected with vector control, wild-type PKD1 or PKD1.CRD. Cells were stimulated with H2O2 (10 mM, 10 minutes) as indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blotting was performed with -pS738/S742. Total PKD1 was detected by stripping and re-probing for PKD1. (D) HeLa cells were transfected with vector control, wild-type PKD1, PKD1.P157G, PKD1.P281G or PKD1.P157G.P281G. Cells were stimulated with H2O2 (10 mM, 10 minutes) as indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blotting was performed with -pS738/S742. Total PKD1 was detected by stripping and re-probing for PKD1. All experiments were performed at least three times and obtained similar results.

View larger version (64K): [in this window] [in a new window]   Fig. 4. Mitochondrial localization and activation of PKD1 is dependent on DAG. (A-D) HeLa cells were transfected with GFP-tagged wild-type PKD1 or GFP-PKD1.P157G.P281G. Cells were stimulated with Rotenone (20 µM, 60 minutes) where indicated. GFP-PKD1 localization was detected using confocal microscopy. (E) Cells were transfected with vector control, HA-tagged wild-type PKD1 or PKD1.P157G.P281G. Cells were stimulated with Rotenone (20 µM, 60 minutes) where indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blots were performed against active PKD1 by staining for activation loop phosphorylations (-pS738/742). Total PKD1 was detected by stripping and re-probing for PKD1. All experiments were performed three times and similar results were obtained.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Mutation of DAG-binding sites blocks tyrosine phosphorylation of PKD1. (A) Cells were transfected with vector control, wild-type PKD1 or PKD1.P157G.P281G, and were stimulated with H2O2 (10 mM, 10 minutes) where indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blots were performed against PKD1 phosphorylated at Y95 (-pY95). Total PKD1 was detected by stripping and re-probing for PKD1. (B) Cells were co-transfected with vector control, PKD1 or PKD1.P157G.P281G and either empty vector or constitutively active Src (Src.Y527F). PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blots were performed with -pY95, re-probed with -pS738/S742 and -PKD1. Immunoblotting was performed against Src (-Src) to control overexpression. All experiments were performed three times and similar results were obtained.

View larger version (32K): [in this window] [in a new window]   Fig. 6. PLD activity is required for oxidative-stress-mediated activation of PKD1. (A) HeLa cells express PLD1 and PLC. Cells were lysed 24 hours after plating and proteins were resolved by SDS-PAGE. Immunoblotting was performed against PLD1, PLD2 and PLC. Equal loading was controlled by immunoblotting against actin (-Actin). (B) PLC and PLD1 activate PKD1. Cells were co-transfected with PKD1 and control vector, PLC or PLD1. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blotting was performed with -pS738/S742, and the blot re-probed with -PKD1 to control expression. (C) Inhibition of pathways of DAG production. Abbreviations: DAG, diacylglycerol; DGK, diacylgylcerol kinase; IP3, inositol 1,4,5-triphosphate; LPA, lysophosphatidic acid; LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; PI 4,5P2, phosphatidylinositol 4,5-bisphosphate; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D. (D) Inhibition of PLC by U73122 has no effect on PKD1 activation in response to oxidative stress. Cells were transfected with PKD1, and either incubated with U73122 (5 µM, 30 minutes) or vehicle control. Cells were stimulated with H2O2 (10 mM, 10 minutes) where indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blotting was performed with -pS738/S742, and the blot re-probed with -PKD1 to control expression. (E,F) Inhibition of PLD blocks PKD1 activation in response to oxidative stress. Cells were transfected with PKD1, and either incubated with 1-butanol (0.2%, 10 minutes; E), propranolol (250 µM, 30 minutes; F) or left untreated. Cells were stimulated with H2O2 (10 mM, 10 minutes) where indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blots were performed with -pS738/742, and the blot re-probed with -PKD1 to control expression. Experiments were performed three times and similar results were obtained each time.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Lipase-deficient PLD1 blocks PKD1 activation in response to ROS. (A,B) Cells were co-transfected with PKD1 and vector control, wild-type PLD1 or PLD1.K898R. Cells were stimulated with Rotenone (20 µM, 1 hour; A) or H2O2 (10 mM, 10 minutes; B) where indicated. PKD1 was immunoprecipitated (-HA) and resolved by SDS-PAGE. Western blots were performed with -pS738/S742. Total PKD1 was detected by stripping and re-probing for PKD1. (C) Cells were co-transfected FLAG-tagged PKD2 or GST-tagged PKD3 with vector control, wild-type PLD1 or PLD1.K898R. Cells were stimulated with H2O2 (10 mM, 10 minutes) where indicated. PKD2 (-FLAG) or PKD3 (-GST) was immunoprecipitated and resolved by SDS-PAGE. Western blots were performed with -pS738/S742. Total PKD1 was detected by stripping and re-probing for PKD1. All experiments were performed three times and similar results were obtained.

View larger version (132K): [in this window] [in a new window]   Fig. 8. Inhibition of PLD1-mediated DAG formation regulates the localization of PKD1 to the mitochondria. (A-P) Cells were seeded on eight-well µ-slides and transfected with GFP-tagged PKD1 and pDsRED2-Mito (mitochondrial marker). Cells were either treated with solvent or treated with U73122 (5 µM, 10 minutes), 1-butanol (0.2%, 10 minutes) or with propranolol (250 µM, 10 minutes) and then stimulated with Rotenone (20 µM, 1 hour). After stimulation, the cells were fixed and analyzed. The experiment was performed three times and similar results were obtained.

View larger version (97K): [in this window] [in a new window]   Fig. 9. PLD1 regulates the localization of PKD1 to the mitochondria. (A-L) Cells were seeded on eight-well µ-slides and transfected with GFP-tagged PKD1, pDsRED2-Mito (mitochondrial marker) and control vector, wild-type PLD1 or PLD1.KR, and then stimulated with Rotenone (20 µM, 1 hour). After stimulation, the cells were fixed and analyzed. The experiments were performed three times and similar results were obtained.

View larger version (76K): [in this window] [in a new window]   Fig. 10. PLD1 regulates the localization of Y463-phosphorylated PKD1 to the mitochondria. (A-L) Cells were transfected with HA-tagged PKD1 or PKD1.Y463E mutant and pDsRED2-Mito (mitochondrial marker). Twenty-four hours after transfection, the cells were seeded on glass coverslips. Cells were either left untreated (A-C,G-I) or stimulated with propranolol (250 µM, 30 minutes) to block DAG formation from PA (D-F,J-L). Immunofluorescence samples were stained and processed as described in the Materials and Methods. Samples were analyzed by confocal microscopy. Green, PKD1 stained with -P8 (-PKD1 antibody); red, pDsRED2-Mito (mitochondria); blue, DAPI (nuclei). (M-P) Cells were seeded on eight-well µ-slides and transfected with GFP-tagged PKD1.Y463E, pDsRED2-Mito (mitochondrial marker) and control vector or PLD1.KR. Twenty-four hours after transfection, the cells were fixed and analyzed. The experiments were performed three times and similar results were obtained.

View larger version (85K): [in this window] [in a new window]   Fig. 11. Src regulates the localization of PKD1 to the mitochondria. (A) Cells were seeded on eight-well µ-slides and transfected with GFP-tagged PKD1, pDsRED2-Mito (mitochondrial marker) and vector control or Src.Y527F. Twenty-four hours after transfection, the cells were fixed and analyzed by immunofluorescence. (B1-B8) Cells were seeded on eight-well µ-slides and transfected with GFP-tagged PKD1 and pDsRED2-Mito (mitochondrial marker). Twenty-four hours after transfection, the cells were pre-treated with PP2 (10 µM, 1 hour) and then stimulated with Rotenone (20 µM, 1 hour) as indicated. Cells were then fixed and analyzed by immunofluorescence. All experiments were performed three times and similar results were obtained.

View larger version (28K): [in this window] [in a new window]   Fig. 12. Mechanisms of sequential PKD1 activation in response to oxidative stress. Oxidative-stress-mediated PLD1 activation leads to an increase in PA and subsequent DAG formation. The initial phosphorylation of PKD1 at Y463 via Src (1) allows the recruitment of PKD1 to the mitochondria via DAG binding (2). Binding leads to a change in conformation, allowing further Src-mediated phosphorylations. Src directly phosphorylates Y95, creating a C2 domain-binding motif (3), to which PKC binds and phosphorylates S738/742 to fully activate PKD1 (4). PKD1 can then activate downstream targets to facilitate mitochondria-to-nucleus signaling.

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