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First published online 19 August 2003
doi: 10.1242/jcs.00700

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Parkin is recruited to the centrosome in response to inhibition of proteasomes

Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA

View larger version (110K): [in a new window]   Fig. 1. Lactacystin-induced accumulation of parkin in the centrosome. SH-SY5Y cells were treated without (-Lac; A-C) or with (+Lac, D-F) lactacystin, and co-stained with antibodies against the centrosome marker -tubulin (-tub; A,D) and parkin (PKN; B,E). Merged images (C,F) showed significant accumulation of parkin in the centrosome after lactacystin treatment (10 µM for 12 hours). The experiment was repeated three times with similar results. Scale bars: 5 µm.

View larger version (94K): [in a new window]   Fig. 2. Recruitment of parkin to the centrosome was dependent on the duration and dosage of lactacystin treatment. Representative confocal images from SHSY5Y cells treated with 10 µM lactacystin for 0, 4, 8 and 12 hours (A-D). Time-dependent increase of parkin (green) was seen in the centrosome, where -tubulin (red) signals overlapped (yellow) with some of parkin signal. The area of the -tubulin staining also increased with time of treatment. Scale bars: 5 µm. (E) The average area of parkin accumulation was plotted against the duration of lactacystin (10 µM) treatment. #P<0.05 vs. 0 hour; *P<0.001 vs. 0 hour (n=19 cells for each point). (F) The average area of parkin accumulation was plotted against the concentration of lactacystin treated for 12 hours. *P<0.001 vs. 0 µM (n=16 cells for each point).

View larger version (158K): [in a new window]   Fig. 3. Lactacystin did not cause a general recruitment of proteins to the centrosome. No significant centrosomal accumulation was seen for CREB (CREB; A,B) or MAP kinase (MAPK; C,D) in SH-SY5Y cells treated without (-Lac; A,C) or with (+Lac; B,D) 10 µM lactacystin for 12 hours. The experiments were repeated three times with similar results. Scale bars: 5 µm.

View larger version (70K): [in a new window]   Fig. 4. Lactacystin-induced recruitment of transfected parkin in HEK293 cells. HEK293 cells were transfected with a parkin expression construct, treated without (-Lac; A,C,E) or with (+Lac; B,D,F) 10 µM lactacystin for 12 hours, and co-stained with antibodies against -tubulin (-tub; A,B) and parkin (PKN; C,D). Merged confocal images (E,F) show that parkin accumulated in the centrosome in some of the transfected cells after lactacystin treatment (indicated by arrows in F). Scale bars: 5 µm. (G) Percentage of transfected cells that have centrosomal accumulation of parkin was plotted against the concentration of the lactacystin applied (12 hours). (H) Percentage of transfected cells that have centrosomal accumulation of parkin was plotted against the duration of lactacystin treatment (10 µM). *P<0.05 vs. 0 µM (G) or 0 hour (H) (n=3 fields of view per data point, with at least 100 transfected cells per field).

View larger version (31K): [in a new window]   Fig. 5. Binding between parkin and -tubulin in the rat brain and HEK293 cells. (A) Ultracentrifuged rat brain homogenates were incubated with or without colchicine (25 µM for 15 minutes at 4°C) and immunoprecipitated with pre-immune serum (Pre) or anti-parkin (PK) that had been pre-incubated with or without its antigenic peptide. Immunoprecipitates and 1% of the input material (In) were analyzed by western blotting (WB) with a monoclonal antibody against -tubulin. The co-immunoprecipitation of -tubulin with parkin was not affected by colchicine, and was abolished when the antibody was pre-absorbed with its antigen. (B) Ultracentrifuged rat brain homogenates treated with or without colchicine were immunoprecipitated with an irrelevant antibody (n, anti-neurabin) or -tubulin antibody (). Precipitated proteins and 1% of the input material (In) were analyzed by western blotting with antibodies against -, ß- or -tubulins, respectively. Only a very small fraction of -tubulin was co-immunoprecipitated with /ß-tubulin heterodimers. (C) Cleared lysates from HEK293 cells transfected with or without FLAG-tagged parkin were immunoprecipitated with anti-FLAG. Precipitated proteins and 3% of input material were analyzed by western blotting with anti--tubulin. The input was also blotted with anti-FLAG to detect the expression of the parkin construct. -tubulin, which was co-immunoprecipitated with parkin, migrated at 50 kDa. IgG HC, heavy chain of IgG. The experiments were repeated at least three times with similar results.

View larger version (122K): [in a new window]   Fig. 6. Colchicine or taxol treatment blocked the centrosomal recruitment of parkin induced by lactacystin. SH-SY5Y cells were treated with colchicine (+Col; A-C), colchicine plus lactacystin (+Col +Lac; D-F), taxol (+Tax, G-I), or taxol plus lactacystin (+Tax +Lac, J-L) for 12 hours, all at 5 µM. They were fixed and co-stained with anti--tubulin (red), anti-parkin (green) and the DNA-binding dye TO-PRO-3 (blue). The experiments were repeated at least three times with similar results. Scale bars: 5 µm.

View larger version (81K): [in a new window]   Fig. 7. Lactacystin, colchicine or taxol treatment did not alter the total amount of parkin expression in the cell. SH-SY5Y cells were treated with vehicle (-), lactacystin (L), taxol (T), colchicine (C), lactacystin plus taxol (L + T), or lactacystin plus colchicine (L + C), all at 5 µM, for 12 hours. Cell lysates were separated by centrifugation at 4°C into supernatant and pellet fractions, and analyzed by western blotting with anti-parkin or anti-p38. Experiments were repeated three times, each with similar results.

View larger version (141K): [in a new window]   Fig. 8. Lactacystin-induced accumulation of CDCrel-1 and ubiquitinated proteins in the centrosome. HEK293 cells transfected with myc-tagged CDCrel-1 were treated without (-Lac; A-C,G-I) or with (+Lac; D-F,J-L) lactacystin (10 µM for 12 hours) and co-stained with antibodies against -tubulin (-tub; A,D) and myc-tag (myc; B,E), or anti--tubulin (-tub; G,J) and anti-ubiquitin (Ub; H,K), respectively. Merged confocal images showed that CDCrel-1 (F and C) and ubiquitinated proteins (L and I) accumulated in the centrosome after lactacystin treatment. Scale bars: 5 µm. Similar results were obtained from at least five sets of confocal images taken from two to three coverslips at each condition. (M) Anti-myc western blot of lysates from cells transfected without (-) or with (+) myc-CDCrel-1 to show expression of the protein and specificity of the antibody (only one band at the correct size was recognized). (N) Anti-ubiquitin western blot of lysates from cells treated with 0 µM, 1 µM, or 10 µM lactacystin (Lac) for 12 hours. Most of the ubiquitin signals represented polyubiquitinated proteins with very little free ubiquitin running at 8.5 kDa (*).

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