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First published online 10 October 2006
doi: 10.1242/jcs.03219

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14-3-3 affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

¹ The Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education and The State Key Laboratory of Bio-membrane and Membrane Bio-engineering, Peking University, Beijing 100871, China
² The Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China
³ The Center for Theoretical Biology, Peking University, Beijing 100871, China
⁴ Neuroscience Research Institute, Peking University, Beijing 100083, China
⁵ Hong Kong DNA Chips Limited, Hong Kong SAR, China

View larger version (61K): [in a new window]   Fig. 1. Association of 14-3-3 and GFAP is independent of vimentin. (A) Immunoprecipitation assay by GFAP antibody in astrocytes. The astrocyte extracts (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or a rabbit anti-GFAP antibody (lane 3) and subsequent western blot analysis with mouse anti-vimentin antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also detected by vimentin and 14-3-3 antibodies (lane 4). (B) Immunoprecipitation assay by vimentin antibody in astrocytes. The astrocyte extracts (lane 1) were subjected to immunoprecipitation with a mus preimmune IgG (lane 2) or a mus anti-vimentin antibody (lane 3) and subsequent western blot analysis with rabbit anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also detected by GFAP and 14-3-3 antibodies (lane 4). (C) Western blot analysis of SW13 Cl1 (lane Cl1) and SW13 Cl2 (lane Cl2) using specific antibodies to vimentin (top) and 14-3-3 (bottom). (D) Immunoprecipitation of 14-3-3 and vimentin. The extracts of SW13 Cl1 cells (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or the rabbit anti-14-3-3 antibody (lane 3) and subsequently detected with mouse anti-vimentin antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also subjected to western blot (lane 4). (E) Results of western blot of pcDNA3.1-GFAP transfected SW13 Cl2 cells. The proteins were detected by mouse anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The same amount of untransfected cells was used as in control (lane control). (F) The interaction of GFAP and 14-3-3 in pcDNA3.1-GFAP transfected SW13 Cl2 cells. SW13 Cl2 extracts transiently expressing GFAP (lane 1) were subjected to immunoprecipitation with rabbit anti-GFAP antibody (lane 3) or a rabbit preimmune IgG (lane 2) and subsequent western blot with mouse anti-GFAP antibody (top) or mouse anti-14-3-3 antibody (bottom). The pellet was also subjected to western blot analysis (lane 4).

View larger version (26K): [in a new window]   Fig. 2. Effect of the hyperphosphorylation of GFAP on the association of GFAP and 14-3-3. (A) GFAP immunoprecipitation from GFAP transfected SW13 Cl2 cells by hyperphosphorylation and dephosphorylation treatments. The cells were treated with serum-deprived medium (lanes 2 and 4) or medium containing 100 nM Calyculin A (lanes 1 and 3). Then the cell lysate was incubated with buffer alone (lanes 1 and 2) or with CIAP (lanes 3 and 4). The immunoprecipitates were blotted with anti-GFAP or anti-14-3-3 antibody. (B) GFAP immunoprecipitation from GFAP transfected SW13 Cl2 cells by kinase treatments. The cell lysate was incubated with beads and GFAP antibody. After centrifugation, GFAP binding complex was incubated with CIAP for 30 minutes and then, was treated with cdc2 kinase (lane 2) or PKC (lane 3) in relative buffer. Then fresh cell lysate was incubated with kinase treated GFAP binding complex and the final immunoprecipitates were blotted with anti-GFAP or anti-14-3-3 antibody.

View larger version (55K): [in a new window]   Fig. 3. Association of 14-3-3 with GFAP during the cell cycle. (A) Coimmunoprecipitation of GFAP and 14-3-3 in synchronized astrocytes using GFAP antibody. The primary astrocytes were blocked at G1/S phase using aphidicolin (lane 1). Then the cells were released from the block for the indicated time (lanes 2-5). (B) Immunofluorescent triple staining of astrocytes in different phases of the cell cycle with GFAP (green), 14-3-3 (red) and DNA (blue). Scale bars, 10 µm.

View larger version (58K): [in a new window]   Fig. 4. Identification of the 14-3-3 interaction domain of GFAP. (A) Schematic presentation of GFAP and its deletion mutants. Like all IF proteins, GFAP contains an N-terminal head domain, a C-terminal tail domain and a conserved -helical rod domain. Two deletion mutants (H and T) of GFAP were constructed by PCR, and c-myc was inserted into the C-terminal of the mutants. (B) The immunoprecipitation assay of c-myc tagged GFAP and its deletion mutants with 14-3-3. SW13 Cl2 cell extracts were subjected to immunoprecipitation with rabbit anti-GFAP antibody, subsequent western blot with the mouse anti-c-myc (upper) and anti-14-3-3 (middle) antibodies. Whole cell lysates were also subjected to western blot by 14-3-3 antibody as the control (bottom). (C) Colocalization of GFAP and 14-3-3. SW13 Cl2 cells were transfected with GFAP and its mutants (H and T). The cells were immunostained by mouse anti-c-myc antibody (green) and rabbit anti-14-3-3 antibody (red). (D) FRET between 14-3-3-CFP and GFAP-YFP in transfected SW13 Cl2 cells. The fluorescent images showed the recordings immediately before and after the photobleaching cycle. The bleaching zone (ROI1) was marked with a red arrow and the control zone (ROI2) was marked with a white arrow. (E) The plot shows the fluorescence intensities in the two regions for each channel in Fig. 4D. (F) FRET efficiencies (Ef) between 14-3-3-CFP and YFP-tagged GFAP deletion mutants. Ef is represented as the differences of CFP fluorescence before and after YFP photobleaching in defined regions (ROI 1, see Fig. 4D). Similar calculations were done in a non-bleached region (ROI 2) of comparable intensity of the same cell to calculate the control value (Cf). In each experiment, n=10 cells were recorded and analyzed per condition. *The differences between the paired Ef and Cf values marked by asterisks are highly significant (P<0.001). The differences between the paired Ef and Cf values marked by # were not significant (P>0.05). Scale bar, 10 µm. Scale bar in magnified figure, 5 µm.

View larger version (46K): [in a new window]   Fig. 5. Requirement of GFAP phosphorylation at Ser8 for GFAP-14-3-3 interaction. (A) Schemes showing the N-terminal amino acid sequences of human and mouse GFAP (upper), and its substitution mutations (below). (B) Representative immunostaining images of GFAP mutants and 14-3-3 in transfected SW13 Cl2 cells. Scale bar, 10 µm. (C) The bar graphs show quantification of the colocalization of GFAP mutants and 14-3-3 in (B). In each experiment, transfected interphase cells (n=100) were recorded and the displayed data are representative of three independent experiments. *The differences between S8A and WT (wide type) values marked by asterisks are highly significant (P<0.001). The differences between other mutants and WT values showed no significance (P>0.05). (D) The immunoprecipitation assay of GFAP and its serine site mutants with 14-3-3. Extracts from mutant transfected SW13 Cl2 cells were subjected to immunoprecipitation with rabbit anti-GFAP antibody, and subsequent western blot with mouse anti-14-3-3 and anti-c-myc antibodies. Whole cell lysates were also detected by mouse 14-3-3 antibody and rabbit GFAP antibody as the control.

View larger version (56K): [in a new window]   Fig. 6. Phenotype of glial filaments by overexpression of 14-3-3. (A) C6 cells were transfected with DsRed-14-3-3 or DsRed, and GFAP filaments were visualized by immunostaining with GFAP antibody. (a) Normal glia filaments immunostained with rabbit GFAP antibody. (b-c) pDsRed-N1 vector transfection (c, red) did not affect GFAP filaments (b). (d-i) In DsRed-14-3-3 overexpressing cells (e,h), endogenous GFAP (d,i) showed an aggregation of dots. Scale bar, 10 µm. (B) Distribution of glial filaments in the population of cells expressing the indicated proteins. A minimum of 100 cells was randomly scored in a blinded manner. Each transfection was repeated three times with similar results.

View larger version (80K): [in a new window]   Fig. 7. Motile properties of GFAP in 14-3-3 overexpressed glioma C6 cells. (A) Cells transfected with GFP-GFAP were imaged before (a,b), after bleaching (c,d), and during recovery (e-h). Images were taken at the indicated times after the end of the bleach pulse. The indicated area is enlarged in pseudocolour (b,d,e-h) and the fluorescence density is shown on the right. (B) Quantitative analysis of FRAP experiments after bleaching. Cells expressing only GFP-GFAP were detected as the control (a). Two different colocalized forms of GFAP, filaments (b) and dots (c), were both examined in GFP-GFAP and DsRed-14-3-3 cotransfected cells. (C) The t1/2 of FRAP in the above three groups were calculated. Scale bar, 10 µm. Scale bar in magnified figure, 1 µm.