Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons - PubMed (original) (raw)
Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons
C Gieffers et al. Proc Natl Acad Sci U S A. 1999.
Abstract
The anaphase-promoting complex/cyclosome (APC) is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1. Recent work has shown that activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1. Here we show that the mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain. Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons. The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1. Purified brain APC(CDH1) has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC(CDC20). On the basis of these results, we propose that the functions of APC(CDH1) are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells.
Figures
Figure 1
Analysis of APC expression in mouse tissues. Equal amounts of protein from 10,000 × g supernatant fractions from different mouse tissue extracts were separated by SDS/PAGE and analyzed by immunoblotting by using antibodies to the indicated proteins. The CDH1 crossreactive band of slower mobility detected in brain extract neither cofractionated nor coimmunoprecipitated with APC (see Fig. 4_B_ and not shown).
Figure 2
Expression of APC in hippocampal neurons. (A) Immunoblot analysis of CDC27 in extracts from rat hippocampal neurons differentiated in vitro for 3 or 9 days and in mouse brain extract. (B) Characterization of monoclonal APC antibodies. Protein extracts from HeLa and MDCK cells and from mouse and cow brain were analyzed by immunoblotting with antibodies APC2–30 and CDC27. (C) Immunofluorescence microscopy of rat hippocampal neurons differentiated in vitro for 1 wk (a–d) and of cultured MDCK cells (e and f), by using APC2–30 (a and f) and CDC27 antibodies (d). b and c show the same neuron as in a stained with DAPI and visualized by phase contrast microscopy, respectively. The cell in c shows the typical morphology of a stage 3–4 neuron (16) with three dendrites that are thick at the base and taper with distance from the cell body, whereas thin axons of uniform diameter (indicated by arrows) are running along the dendrites. Cells in e are the same as in f, stained with DAPI. Size bars = 10 μm.
Figure 3
Expression of APC and CDH1 in adult mouse brain. Paraffin sections (A–F) or frozen sections (G and H) of mouse brain were analyzed by peroxidase immunohistochemistry (A–F) or by immunofluorescence microscopy (G and H). Sections were incubated with antibodies to CDC27 (B), CDC20 (C and E), CDH1 (D and F), APC2 (H), or with secondary antibodies alone (A). G shows the same section as in H stained with DAPI. Sections in A–F were counterstained with hematoxilin/eosin. I and J show cultured mouse EpH4 cells stained with DAPI (I) or with CDC20 antibodies (J). Size bars = 10 μm for G–J, 50 μm for A–F.
Figure 4
Biochemical and enzymatic characterization of brain APCCDH1. (A) Identification of APC subunits by silver staining: immunoprecipitates obtained with affinity-purified rabbit CDC27 antibodies from logarithmically growing HeLa cells or total mouse brain extracts were analyzed by SDS/PAGE and silver staining. The three proteins marked with stars could not reproducibly be precipitated. (B) Immunoblot analysis of cow brain proteins separated by Q-column anion exchange chromatography, by using antibodies to the indicated proteins. As a positive control, proteins from bovine cells enriched in mitosis by nocodazole treatment were analyzed side by side. (C) Brain APC is an active ubiquitin ligase. Immunopurified cow brain APC was used to ubiquitinate radiolabeled cyclin B. Samples taken at the indicated time points were analyzed by SDS/PAGE and PhosphorImaging (Molecular Dynamics). N-terminal recombinant fragments of sea urchin (cyclin B 13–110) and Xenopus (cyclin B 1–102) cyclin B were used as substrates. The corresponding D box mutants 1–102 ΔDb and 13–110 Db-AA were analyzed side by side.
Figure 5
Analysis of the D box dependence of brain APC. (A) Flow cytometric analysis of HeLa cells: HeLa cells were enriched in G1 phase by growth to confluency or arrested in mitosis by treatment with nocodazole, and their DNA contents was analyzed by flow cytometry. (B) Analysis of CDC20 and CDH1 association with APC: APC immunoprecipitates from extracts of confluent and mitotic HeLa cells and from cow brain Q10 fraction were analyzed by immunoblotting with CDC20, CDH1, APC10/DOC1, and CDC27 antibodies. Immunoblots marked with * (CDH1 and APC10) were performed by using an iodinated secondary antibody. (C) Relative ratio of CDH1/APC10 by using the data of the immunoblot shown in B, quantitated by PhosphorImaging. (D) Kinetics of ubiquitination reactions: ubiquitination assays by using immunoprecipitated APC from cow brain, confluent and mitotic HeLa cells were performed with cyclin B 13–110 wild type (wt) or 13–110 Db-AA mutant as described for Fig. 4_C_. The percentage of cyclin B-ubiquitin conjugates was quantitated for each time point. (E) Ratios of ubiquitination activities toward cyclin B Db-AA vs. activities toward wt cyclin B, calculated for the time point at 10 min.
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