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First published online 24 January 2006
doi: 10.1242/jcs.02778

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APRO4 negatively regulates Src tyrosine kinase activity in PC12 cells

INSERM U584, Faculté de Médecine Necker-Enfants Malades, 156 Rue de Vaugirard, 75730 Paris CEDEX 15, France

View larger version (36K): [in a new window]   Fig. 1. In vitro interaction of Flag-APRO4 with Srcwt. (A) The various APRO4 mutants used are represented schematically and the amino-acid sequence of the C-terminal region of APRO4 (145-152) is also shown. Proline residues that were mutated to alanine are represented on the amino-acid sequence. (B, left panels) In vitro translated 35S-labelled Flag-APRO4, and X-APRO4N associated with Src GST-SH3-SH2 whereas Flag-APRO4C did not. (C, left panel) Flag-APRO4 interacted with Fyn SH3-SH2 domains but not with Lyn, Blk, and various GST-SH3-SH2 fusion proteins: GAP (GTPase activating protein), PLC (phospholipase C). (B,C, right panels) Coomassie staining of SDS-PAGE of purified GST-fusion proteins. Input: 100% of the respective 35S-labelled proteins used in binding reactions; GST: GST-beads. Data are representative of three independent experiments.

View larger version (26K): [in a new window]   Fig. 2. In vivo association of APRO4 with Src. (A-D) NIH3T3 cells were transiently transfected with the described constructs and analysed by co-immunoprecipitation (IP) followed by immunoblotting (IB) with anti-Src, anti-Flag or anti-Xpress (anti-X). X-APRO4N interacted with Srcwt (A) whereas Flag-APRO4C did not (B). (C,D, left panels) Co-immunoprecipitation of Srcwt, K297M, Y529F, K297M Y529F, and W118R R175K with Flag-APRO4 in NIH 3T3 cells co-expressing Src constructs and Flag-APRO4. (C,D, right panels) Co-immunoprecipitation of Srcwt, Src W118R, Src R175K, and Src W118R R175K with Flag-APRO4 or Srcwt with Flag-APRO4-6PA in NIH 3T3 cells co-expressing the described constructs. Note that no IgG band was detected on the Src immunoblot of the anti-Flag immunoprecipitates (C, top right panel) because a polyclonal anti-Src (SRC2) antibody was used. This antibody, unlike the monoclonal anti-Src antibody (MAb 327) that was used in all other figures, does not cross react with the heavy IgG chain. Whole-cell lysates of transfected cells were analysed by western blotting with the appropriate antibody to verify equal expression of all the constructs. Data are representative of three independent experiments.

View larger version (39K): [in a new window]   Fig. 3. Interaction of endogenous APRO4 with endogenous Src in PC12 cells. (A) Co-immunoprecipitation of endogenous APRO4 with endogenous Src in PC12 cells. The normal mouse antiserum did not immunoprecipitate APRO4. (B) Subcellular localization of endogenous Src and APRO4 in PC12 cells by confocal microscopy. Cells were stained with anti-Src (green) and anti-APRO4 (red). APRO4 co-localized with Src in the cytoplasm (yellow). Bars, 10 µm.

View larger version (19K): [in a new window]   Fig. 4. Inhibition of Src kinase activity in vitro. (A) Coomassie staining of SDS-PAGE of purified GST-fusion proteins. (B,D) In vitro protein kinase assay were performed with 5 units of purified Src together with 5 µM GST, 0.5-2.0 µM GST-APRO4 or 5 µM of GST-APRO4C and enolase. Enolase was used as an exogenous substrate for measuring Src activity. 32P-labelled proteins were resolved by SDS-PAGE and visualized by autoradiography. Data are representative of four independent experiments. (C,E) Quantification of data obtained in (B) and (D). The incorporation of 32P into enolase was quantified by scanning densitometry. Data represent average values from four independent experiments and are expressed relative to those for purified Src with the addition of GST alone. Error bars indicate standard errors.

View larger version (35K): [in a new window]   Fig. 5. Inhibition of Src kinase activity, Ras/MAP kinase signalling in PC12 cells by overexpression of APRO4. (A,B) PC12 cells transfected with a control empty vector or increasing amounts of APRO4, APRO4N, or APRO4C vectors were stimulated with FGF (25 ng/ml), and assayed for endogenous Src kinase activity. Proteins were immunoprecipitated from lysates containing 1 mg of total cellular protein with an excess of Src MAb 327 followed by in vitro kinase assay (A) or protein immunoblotting with anti-phospho-Y416 and anti-Src (B). Immunoblotting with Src antibody confirmed that equivalent amounts of Src were present in immunoprecipitates reactions. Data are representative of four independent experiments. (C) PC12 cells were co-transfected with 1 µg 5x-Gal4-TATA/luciferase, 200 ng Gal4-Elk1 (Elk/gal), 1 µg CMV-ßGal and the indicated expression plasmids, and either left unstimulated or stimulated with FGF (25 ng/ml) for 6 hours. Activation of Elk1 is reflected by luciferase activity normalized to ßGal activity and is described as fold decrease. Values are presented as the mean ± s.e.m. (error bars) of three independent experiments carried out in triplicate. Differences between two means with a P<0.05 were regarded as significant.

View larger version (68K): [in a new window]   Fig. 6. Inhibition of FGF-mediated PC12 neurite formation by overexpression of APRO4. PC12 cells were transfected with either a control empty vector (data not shown), Flag-APRO4 (A), X-APRO4N (B), Flag-APRO4C (C), or Flag-APRO4 and HA-MEK1 S218/222D (D), stimulated with FGF (25 ng/ml) for 2 days, fixed and immunostained with the indicated antibodies. FGF-stimulated transfected PC12 cells expressing Flag-APRO4 (panel A, white arrows) or X-APRO4N (panel B, white arrows) did not extend neurites whereas Flag-APRO4C transfected PC12 cells did (panel C, white arrows). Untransfected PC12 cells stimulated with FGF developed neurites (panels A-C, black arrows). In cells co-transfected with Flag-APRO4 and HA-MEK1 S218/222D, cells expressing only Flag-APRO4 (red) did not extend neurites (panel D, white arrows) whereas cells expressing Flag-APRO4 (red) as well as HA-MEK1 S218/222D (green) expression plasmids showed extensive neurite outgrowth (panel D, black arrows). Polyclonal anti-Flag antibody, monoclonal anti-Xpress and anti-HA antibodies showed no signal with untransfected PC12 cells (panels A-C, black arrows). More than 300 transfected PC12 cells were analysed per condition in four independent experiments. Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 7. Inhibition of FGF-mediated PC12 cell differentiation by overexpression of APRO4. (A,B) PC12 cells were transiently transfected with either an empty control vector, Flag-APRO4, X-APRO4N, Flag-APRO4C, or Flag-APRO4 and the indicated expression plasmids. After 24 hours, cells were serum starved for 20 hours, then either left untreated or treated with FGF for 2 days and analysed for expression of the differentiation marker Tubulin-ßIII by immunoblotting (IB). Actin was used as an internal control to verify equal loading of proteins. Data are representative of three independent experiments.

View larger version (27K): [in a new window]   Fig. 8. Downregulation of Src kinase activity correlated with increased endogenous APRO4 expression in FGF-stimulated PC12 cells. (A) Upregulation of endogenous APRO4 protein expression in FGF stimulated cells. PC12 cells were serum starved for 20 hours and then stimulated with FGF (25 ng/ml) for the indicated times before being collected. Whole-cell lysates were immunoblotted with the indicated antibodies. Actin was used as internal control to verify equal loading of proteins in each condition. Densitometric quantitation of relative APRO4 expression is indicated underneath each lane of the top panel. (B) Serum starved PC12 cells were stimulated with FGF (25 ng/ml) for the indicated times, and analysed by co-immunoprecipitation (IP) followed by immunoblotting (IB) with anti-phospho-Y416, anti-Src and anti-APRO4. Immunoblotting with Src antibody confirmed that equivalent amounts of Src were present in immunoprecipitates reactions. Densitometric quantitation of relative Src phosphorylation is indicated underneath each lane of the top panel. Data are representative of three independent experiments.

View larger version (49K): [in a new window]   Fig. 9. Activation of Ras/MAP kinase signalling and formation of neurites by downregulation of endogenous APRO4 in PC12 cells. (A) Downregulation of endogenous APRO4 by antisense vector, pAS-APRO4. PC12 cells were transiently transfected with a control empty vector or pAS-APRO4, and 200 µg of whole-cell lysates were immunoblotted with the indicated antibodies. (B) Y416 phosphorylation was increased in PC12 cells transiently transfected with pAS-APRO4 compared to the control transfected cells whereas Y416 phosphorylation was inhibited when PC12 transfected cells were treated with 2.5 µM of PP1. Data are representative of four independent experiments. (C) Activation of Elk1 dependent transcription by inhibition of endogenous APRO4 expression. PC12 cells were transiently co-transfected with 1 µg of 5x-Gal4-TATA/luciferase, 200 ng of Gal4-Elk1 (Elk/gal), 1 µg of CMV-ßGal, and pAS-APRO4 or a control empty vector. Activation of Elk1 reflected by luciferase activity and normalized to ßGal activity is described as fold increase. Values are presented as the mean ± s.e.m. (error bars) of three independent experiments carried out in triplicate. (D) Increased ERK phosphorylation in PC12 cells transiently transfected with pAS-APRO4 and inhibition of ERK phosphorylation by PP1. Immunoblotting with ERK antibody confirmed that equivalent amounts of ERK were present in whole-cell lysates of transfected cells. Data are representative of three independent experiments. (E) Induction of PC12 cell neurite outgrowth by inhibition of endogenous APRO4 expression. Cells were transfected with a control empty vector or pAS-APRO4 together with pEGFP-N3 vector. This latter vector was used to positively identify transfected cells. The procedure was checked for immunostaining, showing 80-90% concordance in expression of concomitantly transfected plasmids (data not shown). Transfected cells were then either left untreated or treated with 2.5 µM PP1, fixed after 3 days and immunostained with anti-APRO4 antibody. Processes greater than two diameters of the cell body were defined as neurites. In cells positively transfected with pAS-APRO4, as revealed by GFP staining, endogenous APRO4 was not detected by immunostaining with anti-APRO4 antibody (top and bottom panels, white arrows) whereas in untransfected cells, endogenous APRO4 could be detected (top and bottom panels, black arrows). Light microscopy observation showed that pAS-APRO4 transfected cells developed extensive neurites (top left panel, white arrows) whereas untransfected cells did not (top left panel, black arrows). pAS-APRO4 transfected PC12 cells (bottom left panel, white arrows) as well as untransfected cells (bottom left panel, black arrows) did not develop neurites in the presence of the Src inhibitor PP1. More than 300 pAS-APRO4 transfected PC12 cells were analysed in four independent experiments. Bars, 10 µm.

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