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First published online March 4, 2009
doi: 10.1242/10.1242/jcs.038109

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βPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells

Département Neurotransmission et Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives (UPR 3212), Centre National de la Recherche Scientifique et Université de Strasbourg, 5 rue Blaise Pascal, 67084 Strasbourg, France

View larger version (26K): [in this window] [in a new window]   Fig. 1. Rac1 is localized at the plasma membrane and activated in secretagogue-stimulated PC12 cells. (A) GTP-loaded Rac1 pull-down assay in resting and K+-stimulated PC12 cells. PC12 cells were stimulated with 59 mM K+ for the indicated period of time or maintained under resting conditions in Locke's solution (K+ 59 mM; 0 minutes). Cells were then immediately lysed and lysates (500 µg of proteins) were used for affinity precipitation of GTP-loaded Rac. Pulled down Rac1-GTP and Rac1 in lysates (1/50 of the total) were detected by immunoblotting using anti-Rac1 antibodies. (B) Histogram illustrating a semi-quantitative analysis of Rac1 activation upon cell stimulation. Values obtained by scanning densitometry analysis are given as the mean values±s.d. (n=3). (C) Quantitative colorimetric assay for Rac activity. Cells were washed and subsequently maintained under resting conditions for 10 minutes in Locke's solution (R) or stimulated for 10 minutes with 59 mM K+ (S). The amount of GTP-loaded Rac in each condition was estimated using an ELISA (n=3). ***P<0.001 compared with resting cells (ANOVA). (D) Time course of K+-evoked GH secretion. PC12 cells were washed in Locke's solution and then stimulated with 59 mM K+ for various periods of time. Levels of GH secreted into the medium and retained in cells were estimated using an ELISA. GH release is expressed as the percentage of total GH present in the cells before the stimulation period (n=3). (E) Localization of endogenous Rac1 in PC12 cells. Confocal immunofluorescence images obtained by labelling PC12 cells with monoclonal anti-Rac1 antibodies visualized with Alexa 555-conjugated secondary antibodies and polyclonal anti-SNAP25 antibodies visualized with Alexa 488-conjugated secondary antibodies. The mask obtained by selecting double-labelled pixels represents the regions of Rac1/SNAP25 colocalization. Bar, 5 µm. (F) Histogram representing a semi-quantitative analysis of the percentage of Rac1 signal colocalized with SNAP25 in resting (R) and stimulated (S) cells. Data are given as the mean values±s.d. (n=25 cells) obtained in three independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Reduction of endogenous Rac1 by RNA interference inhibits GH secretion from PC12 cells. PC12 cells were transfected with the pGHsuper vector (control) or with pGHsuper-UnR siRNA or the pGHsuper-Rac1 siRNA plasmids for 72 hours. (A) Efficiency of the Rac1 siRNA. Cells were lysed and aliquots (20 µg of proteins) were used for electrophoresis and western blot analysis using antibodies against Rac1, RhoA, Cdc42 and actin. (B) Assay for GH secretory activity in cells expressing Rac1 siRNA. Transfected cells were incubated for 10 minutes in Locke's solution (resting) or stimulated for 10 minutes with 59 mM K+ (K+-stimulated). GH release was estimated from resting and K+-stimulated PC12 cells transfected with the indicated plasmid (n=3). ***P<0.001, NS (not significant) compared with control cells (ANOVA). (C) Rescue of GH secretion in Rac1-depleted PC12 cells. Cells were co-transfected with pCDNA3-HA vector or with the plasmid encoding Ha-tagged human Rac1 (hRac1) along with the pGHsuper encoding the shRNA for Rac1 (Rac1 siRNA). Control cells were co-transfected with the pGHsuper and pCDNA3-HA vectors. Cells were stimulated for 10 minutes with 59 mM K+ and processed for GH release assay (n=3). ***P<0.001, NS (not significant) compared with control cells (ANOVA).

View larger version (34K): [in this window] [in a new window]   Fig. 3. Rac1 specifically regulates secretagogue-evoked PLD1 activity in PC12 cells. (A) Efficiency of the RhoA siRNA. PC12 cells were transfected with pEGFP-RNAi (control), pEGFP-UnR siRNA or pEGFP-RhoA siRNA plasmids for 72 hours. Proteins were extracted and 20 µg was analysed by immunoblotting using the indicated antibodies. (B) Effect of Rac1, RhoA or Cdc42 depletion on K+-stimulated PLD1 activity. Cells were transfected with the pEGFP vector (Control) or with the plasmids encoding the indicated shRNA. Seventy-two hours after transfection, cells were stimulated for 10 minutes with 59 mM K+ or maintained under resting condition and processed for PLD activity assay. K+-evoked PLD activity is obtained by subtracting the PLD activity detected in resting cells maintained in Ca2+-free Locke's solution from the PLD activity measured in the K+-stimulated cells. PLD activity measured in resting cells remained unchanged between 8 and 10 mU/well. n=4, ***P<0.001, NS (not significant) compared with control cells (ANOVA). (C) Effect of the reduction of endogenous Rac1 on the formation of phosphatidic acid at the plasma membrane in secretagogue-stimulated PC12 cells. Cells transfected with the pGHsuper vector (control) or the pGHsuper-Rac1 siRNA plasmid were plated on four-well plates and transfected again, 48 hours later, with the wtPABD-EGFP plasmid. Twenty-four hours after the second transfection, cells were stimulated for 10 minutes with 59 mM K+ or incubated for 10 minutes in Locke's solution (resting). The intracellular localization of GH (used to identify cells expressing siRNA) and SNAP25 was determined by confocal microscopy analysis using polyclonal anti-GH and monoclonal anti-SNAP25 antibodies visualized with Alexa 647- and Alexa 555-conjugated secondary antibodies, respectively. Masks were obtained by selecting double-labelled pixels as representing the regions of wtPABD/SNAP25 colocalization. Bar, 5 µm. (D) Semi-quantitative analysis of the percentage of wtPABD signal co-localized with SNAP25 in resting or stimulated PC12 cells. Data are given as the mean values±s.d. (n=25 cells) obtained in three independent experiments. ***P<0.001 compared with control cells (ANOVA).

View larger version (29K): [in this window] [in a new window]   Fig. 4. siRNA-mediated βPix knockdown inhibits GH release and reduces secretagogue-evoked Rac1 and PLD1 activation in PC12 cells. PC12 cells were transfected with the pGHsuper vector (control) or with pGHsuper-βPix siRNA plasmid for 48 hours. (A) Efficiency of the βPix siRNA. Cells were scrapped and aliquots (50 µg of proteins) were used for immunoblotting analysis using polyclonal anti-βPix antibodies and monoclonal anti-actin antibodies. (B) βPix siRNA expression inhibits GH secretion. GH release was estimated from resting and K+-stimulated PC12 cells transfected with the indicated plasmid (n=3). ***P<0.001 compared with control cells (ANOVA). (C) Effect of βPix siRNA expression on PLD activity. PC12 cells were transfected with pEGFP vector (control) or pEGFP-βPix siRNA vector. Forty-eight hours after transfection, PLD activity was assayed in resting or K+-stimulated cells. Data are given as the mean values±s.d. (n=6) and normalized for transfection efficiency. **P<0.01 compared with control cells (ANOVA). (D) Effect of βPix siRNA expression on Rac1 activation. PC12 cells co-transfected with pCDNA3Ha-Rac1 and either pGHsuper vector (control) or pGHsuper-βPix siRNA plasmid were stimulated with 59 mM K+ (K+-stimulated) for 10 minutes and lysates (500 µg of proteins) were used for affinity precipitation of GTP-loaded Rac1. The histogram represents a semi-quantitative analysis of Ha-Rac1 GTP detected in resting and K+-stimulated cells. Net Rac1 activation was obtained by subtracting Rac1-GTP level measured in resting cells from the level detected in K+-stimulated cells. Values obtained by scanning densitometry analysis are given as the mean values±s.d. (n=3). **P<0.01, ***P<0.001 compared with control cells (ANOVA).

View larger version (28K): [in this window] [in a new window]   Fig. 5. βPix catalyses the activation of Rac1 and PLD at the plasma membrane in stimulated PC12 cells. (A) βPix translocates to a membrane-bound compartment in stimulated permeabilized PC12 cells. Resting and stimulated PC12 cells were washed and permeabilized with 0.05% saponin in the presence (S) or absence (R) of 20 µM free Ca2+. Soluble proteins (Sol) leaking from cells were collected. Membrane-bound proteins (Mb) retained within cells were solubilized with Triton X-100 and clarified by centrifugation. For each condition, 15 µg proteins per lane were analyzed by western blot with βPix antibodies. (B) Histogram illustrating the relative amount of soluble versus membrane-bound βPix detected in permeabilized resting and stimulated PC12 cells. (C) Localization of Rac1 and βPix in PC12 cells. Cells expressing Flag-βPix were labelled with monoclonal anti-Rac1 and polyclonal anti-βPix antibodies, and analyzed by confocal microscopy. Rac1 and βPix antibodies are visualized with Alexa 555- and Alexa 488-conjugated secondary antibodies, respectively. Bars, 5 µm. (D,E) Effect of a GEF-dead βPix mutant on Rac1 and PLD activation. PC12 cells were transfected with pCDNA3-Ha vector (control), wild-type flag-βPix or flag-βPixL238R-L239S. Rac1-GTP levels (estimated by ELISA) (D) and PLD activity (E) were estimated in resting and K+-stimulated cells (n=3). ***P<0.001, NS (not significant) compared with control cells (ANOVA).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Scrib is required for βPix-mediated activation of the Rac1-dependent pathway. PC12 cells were transfected with the pGHsuper (control) or with pGHsuper-Scrib siRNA plasmid for 48 hours. (A) Silencing efficiency of Scrib siRNA on the level of endogenous Scrib, as analyzed by immunoblotting. (B) Transfected cells were stimulated with high K+ and processed for GH secretion measurement (n=3). ***P<0.001 compared with control cells (ANOVA). (C) GTP-loaded Rac1 pull-down assay in resting and stimulated PC12 cells co-expressing Ha-Rac1 with either, Scrib wild type, Scrib siRNA or ScribPDZ. Control cells were co-transfected with pCDNA3-HA-Rac1 and empty pSuper vectors. The histogram represents a semi-quantitative analysis of Ha-Rac1 activation in resting or high K+-stimulated cells. Values obtained by scanning densitometry analysis are given as the mean values±s.d. (n=3). ***P<0.001, NS (not significant) compared with control cells (ANOVA). (D) Cells transfected with pEGFP vector (control), pEGFP-Scrib wild-type, pEGFP-Scrib siRNA or pEGFP-ScribPDZ plasmids were maintained under resting conditions or stimulated with high K+, and then processed for PLD activity measurement (n=3). NS (not significant), **P<0.01, ***P<0.001 compared with control cells (ANOVA). (E) Distribution of Flag-βPix in resting and K+-stimulated PC12 cells expressing GFP-ScribPDZ. Cells were labelled with polyclonal anti-βPix antibodies and analyzed by confocal microscopy. βPix antibodies are visualized with Alexa 555-conjugated secondary antibodies. Bars, 5 µm. (F) Relative amount of soluble versus membrane-bound βPix present in resting or stimulated ScribPDZ-expressing PC12 cells estimated by cell permeabilization with saponin. Cells were co-transfected with Flag-βPix along with pEGFP-ScribPDZ. Forty-eight hours after transfection, resting and stimulated PC12 cells were permeabilized with 0.05% saponin in the presence or absence of 20 µM free Ca2+, respectively. Cytosolic proteins leaking out the cells (Sol) and membrane-bound proteins (Mb) were analyzed by western blotting scanning densitometry using Flag antibodies to detect βPix.

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