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First published online 7 August 2007
doi: 10.1242/jcs.009613

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Calcineurin is essential for depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2 in neurons

Camille Faure^1,2,3,*, Jean-Christophe Corvol^1,2,3,*, Madeleine Toutant^1,2,3,*, Emmanuel Valjent^1,2,3, Øivind Hvalby⁴, Vidar Jensen⁴, Said El Messari^1,2,3, Jean-Marc Corsi^1,2,3, Gress Kadaré^1,2,3 and Jean-Antoine Girault^1,2,3,

¹ Inserm, U839, Paris F-75005, France
² Université Pierre et Marie Curie, Paris F-75005, France
³ Institut du Fer à Moulin, Paris F-75005, France
⁴ Molecular Neurobiology Research Group, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway

View larger version (35K): [in this window] [in a new window]   Fig. 1. Depolarization induces nuclear translocation of PYK2 in hippocampal slices, independently of Src-family kinases. (A) Rat hippocampal slices incubated in physiological conditions were placed for 2 minutes in high [K+]o (High K+, 40 mM) or normal [K+]o (Control) and immediately fixed. Slices were cut into 30-µm thick sections and stained with anti-PYK2 antibody and DAPI. Bars, 50 µm. (B) Quantification of the percentage of cells with PYK2-positive nuclei. Values are means ± s.e.m., six slices per condition, Student's t-test: ***P<0.001. (C) Time course of the effects of high [K+]o on nuclear localization of PYK2 immunoreactivity. High K+ or control solution was added between 0 and 2 minutes (horizontal bar) and then replaced by standard ACSF. Slices were fixed at the indicated times. Values are means ± s.e.m., two to five slices per data point. Two-way ANOVA: interaction between time and treatment F(3,28)=18.3, P<0.001, treatment effect F(1,28)=29.5, P<0.001, time effect F(3,28)=15.4, P<0.001. Point by point comparison with controls using the Bonferroni test: ***P<0.001. (D) Rat hippocampal slices were treated with vehicle or high [K+]o for 2 minutes in the absence or presence of a Src-family inhibitor, PP2 (20 µM, added 30 minutes before depolarization). Slice homogenates were blotted with a phosphotyrosine antibody (pY, top panel) or with an anti-phospho-Tyr418-Src antibody (p-Src, bottom panel). The positions of PYK2 (110 kDa, p110-PYK2) and Src (60 kDa, p60Src) are indicated. (E) Rat hippocampal slices were treated as in D and PYK2 localization analyzed by immunofluorescence. Bar, 50 µm. (F) Quantification of the percentage of cells with PYK2-positive nuclei in slices treated as in E. Values are means ± s.e.m., five to eight slices per group, one-way ANOVA: F(2,17)=49.1, P<0.001. Newman-Keuls test: ***P<0.001 compared with control.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Tetanic stimulation induces nuclear translocation of PYK2 in hippocampal slices. (A) Mouse hippocampal slices were incubated in physiological ACSF (Control) or treated with high [K+]o (High K+) as in Fig. 1, or subjected to a tetanic stimulation of the stratum radiatum (Schaeffer collaterals, 100 Hz, 4x 1 second, at 10-second intervals). Slices were prepared as in Fig. 1 and stained with an anti-PYK2 antibody or a phospho-specific antibody directed against pY402-PYK2. The CA1 region was analyzed by confocal microscopy (single optical sections are shown). Bars, 10 µm. (B) Quantification of the cells in which anti-PYK2 immunostaining in the nucleus (nuclear PYK2) was equal to or greater than in the cytoplasm. Values are means ± s.e.m., 12 slices per group, one-way ANOVA: F(2,33)=11.2, P<0.001. Newman-Keuls test: **P<0.01, ***P<0.001. (C) Tetanic stimulation of excitatory CA3-to-CA1 synapses potentiated synaptic transmission. Graph shows an example from one of the radiatum pathways. The slopes of the elicited field EPSPs were normalized to the mean value obtained 1 minute prior to tetanization (solid arrows). The slice was removed from the recording chamber 5 minutes after the repeated tetanization procedure and fixed. Inset: superimposed synaptic responses at times indicated by open arrows and numbers.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Depolarization induces Ca2+-dependent nuclear translocation of PYK2 in hippocampal neurons in culture. (A) Rat hippocampal neurons in culture treated with high [K+]o (High K+, 40 mM) or with a control solution for 3 minutes in the presence or absence of EGTA (5 mM, added 5 minutes before depolarization) and stained with an anti-PYK2 antibody were analyzed by fluorescence microscopy. Bars, 5 µm. (B) Quantification of the percentage of cells with nuclear PYK2. Values are means ± s.e.m. (six coverslips per group). Two-way ANOVA: interaction between depolarization and EGTA treatment F(1,20)=48.35, P<0.0001, depolarization effect F(3,20)=62.12, P<0.0001, EGTA treatment effect F(1,20)=77.28, P<0.0001. Newman-Keuls test: PYK2 control vs K+, ***P<0.001, PYK2 K+ vs (PYK2 + EGTA K+), °°°P<0.001. (C) Rat hippocampal neurons in culture treated with high [K+]o (High K+, 40 mM) or with a control solution for 3 minutes and stained with an anti-PYK2 antibody were analyzed by confocal microscopy. A single optical section is shown. Bars, 5 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 4. Depolarization induces nuclear accumulation of PYK2 in PC12 cells. (A) PC12 cells were treated with high [K+]o for 3 minutes or 11 ng/ml leptomycin B for 3 hours. Endogenous PYK2 was detected with an anti-PYK2 antibody and nuclei were stained with DAPI. Bars, 10 µm. (B) Percentage of cells with PYK2 immunoreactivity in the nucleus that was less than (n<c), equal to (n=c) or greater than (n>c) that in the cytoplasm. Values are means ± s.e.m., 2=44.9, d.f.=4, P<0.0001, control vs K+ P<0.0001, control vs lepto B P<0.1, K+ vs lepto B P<0.0001. (C) Cells were immunostained with a pY402-PYK2 phospho-specific antibody and analyzed with confocal microscopy. Bars, 10 µm. (D) Quantification as in B was done for pTyr402-PYK2 immunoreactivity. Values are means ± s.e.m. 2=623.2, d.f.=4, P<0.0001.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Depolarization induces Ca2+-dependent nuclear translocation of GFP-PYK2 in PC12 cells. (A) PC12 cells transfected with GFP-PYK2 were treated with high [K+]o (High K+) or control solution for 3 minutes in the presence or absence of EGTA (5 mM, added 5 minutes before depolarization). GFP-PYK2 fluorescence and nuclear staining with DAPI were analyzed by fluorescence microscopy. Bars, 10 µm. (B) Confocal sections showing the nuclear localization of PYK2. Bars, 10 µm. (C) Quantification of the percentage of cells with nuclear GFP-PYK2 (i.e. fluorescence in the nucleus that was stronger than or equal to that in the cytoplasm). Values are means ± s.e.m. (three to four coverslips per group). Two-way ANOVA: interaction between depolarization and EGTA treatment F(1,11)=10.2, P<0.01, depolarization effect F(1,11)=16.2, P<0.005, EGTA treatment effect F(1,11)=5.5, P<0.05. Newman-Keuls test: PYK2 control vs K+, **P<0.01, PYK2 K+ vs (PYK2 + EGTA K+), °°P<0.01.

View larger version (65K): [in this window] [in a new window]   Fig. 6. PYK2 redistribution in PC12 cells is independent of its autophosphorylation and kinase activity. (A) PC12 cells were transfected with wild-type GFP-PYK2 (WT, lanes 1, 2), GFP-PYK2 Y402F (Y402F, lanes 3, 4) or kinase-dead mutant K457A lanes (K457A, lanes 5, 6), and treated with control or depolarizing solution (K+ 40 mM) for 3 minutes. PYK2 was analyzed by immunoblotting (100 µg protein) with anti-pY402 antibody (upper panel) or with anti-PYK2 antibody (lower panel). (B) PC12 cells transfected with GFP-PYK2 WT, Y402F or K457A were treated with high [K+]o or control solution for 3 minutes. PYK2 immunoreactivity was analyzed by fluorescence microscopy. Bar, 10 µm. (C) Quantification of the percentage of cells with nuclear GFP-PYK2. Values are means ± s.e.m. (four coverslips per group). Two-way ANOVA: interaction between mutation and depolarization F(2,6)=2.1, P>0.05, depolarization effect F(1,6)=72.7, P<0.0001, mutation effect F(2,6)=1.1, P>0.05. Newman-Keuls test: WT control vs K+, **P<0.01, Y402F control vs K+, *P<0.05, K457A control vs K+, **P<0.01. (D) Homogenates (70 µg protein) from control or K+-treated PC12 cells were loaded on a 7% acrylamide gel. Note the downward shift induced by depolarization.

View larger version (44K): [in this window] [in a new window]   Fig. 7. Depolarization-induced phosphorylation and nuclear translocation of PYK2 are abolished by cyclosporin A (CsA) and FK506. (A) PC12 cells were treated with high [K+]o (High K+ 40 mM) or control solution for 3 minutes in the absence or presence of cyclosporin A (10 µM, added 30 minutes prior to depolarization). Endogenous PYK2 was immunoprecipitated and analyzed by immunoblotting with anti-P-Tyr antibody (pY, upper panel). Whole cell lysates were analyzed by immunoblotting with PYK2 antibody (lower panel). (B) Quantification of pY-PYK2 in A. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=65.8, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (CsA + K+) vs K+, °°°P<0.001. (C) PC12 cells transfected with GFP-PYK2 were treated as in A and PYK2 immunofluorescence was analyzed by fluorescence microscopy. Bars, 10 µm. (D) Quantification of results in C. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=18.8, P<0.05. Newman-Keuls test: control vs K+, *P<0.05, (CsA + K+) vs K+, °P<0.05. (E) PC12 cells were treated as in A, except that FK506 (FK506 1 µM, added 30 minutes prior to depolarization) was used instead of cyclosporin A. Endogenous PYK2 was immunoprecipitated and analyzed by immunoblotting with anti-P-Tyr antibody (pY, upper panel). Whole cell lysates were analyzed by immunoblotting for pY402-PYK2 (middle panel) or PYK2 (lower panel). (F) Quantification of pY-PYK2 in E. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=153.1, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001. (G) Quantification of pY402-PYK2 in E. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=162.2, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001. (H) PC12 cells transfected with GFP-PYK2 were treated as in E and PYK2 immunofluorescence was analyzed by fluorescence microscopy. Bars, 10 µm. (I) Quantification of results in H. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=166.2, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Depolarization-induced tyrosine phosphorylation of PYK2 is prevented by a dominant negative form of calcineurin. (A) PC12 cells transfected with GFP-PYK2 in the absence or presence of Flag-PD-CnA (phosphatase-dead calcineurin A) were treated with a depolarizing (High K+ 40 mM; +) or control (–) solution for 3 minutes. PYK2 tyrosine phosphorylation was analyzed by immunoblotting with an anti-P-Tyr antibody (pY, upper panel) and with an anti-PYK2 antibody (middle panel). The presence of Flag-PD-CnA was detected by Flag immunoblotting (lower panel). (B) Quantification of pY-PYK2 in A. Values are means ± s.e.m., of six cultures per group in two independent experiments. Two-way ANOVA: interaction between depolarization and PD-CnA co-transfection F(1,16)=5.3, P<0.05, depolarization effect F(1,16)=8.5, P<0.05, PD-CnA co-transfection effect F(1,16)=5.5, P<0.01. Newman-Keuls test: control vs K+, **P<0.01, PD-CnA K+ vs K+, °°°P<0.001.

View larger version (46K): [in this window] [in a new window]   Fig. 9. The role of calcineurin in nuclear translocation of PYK2. (A) PC12 cells were transfected with a tagged control protein (Flag-protein, FP), a phosphatase-dead form of calcineurin A (Flag-PD-CnA), or a constitutively active calcineurin A (Flag-CA-CnA) and treated for 3 minutes with a control or depolarizing (High K+, 40 mM) solution. Immunofluorescence was analyzed for Flag (left), PYK2 (middle) and merged with DAPI (right). Bars, 10 µm. (B) Control of expression levels of Flag-PD-CnA or Flag-CA-CnA by immunoblotting with an anti-Flag antibody. (C) Quantification of results in A. Values are means ± s.e.m. (four to six per group). Two-way ANOVA: interaction between depolarization and transfection F(2,26)=14.6, P<0.0001, depolarization effect F(1,26)=44.5, P<0.0001, transfection effect F(1,26)=26.3, P<0.0001. Newman-Keuls test: FP control vs FP K+, ***P<0.001, PD-CnA K+ vs FP K+, P<0.001, CA-CnA control vs FP control, °°P<0.01, CA-CnA K+ vs FP K+, P<0.01.