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First published online 9 December 2008
doi: 10.1242/jcs.024554

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CDK phosphorylation of the discs large tumour suppressor controls its localisation and stability

Tumour Virology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

View larger version (28K): [in this window] [in a new window]   Fig. 1. Changes in the pattern of DLG1 expression during the cell cycle. (A) Immunofluorescence analysis of DLG1 expression was performed on HaCaT cells using the anti-DLG1 monoclonal antibody 2D11. Various localisations of the protein can be seen in the three examples, including at sites of cell contact, diffuse cytoplasmic, nuclear excluded and also localisation to the mitotic spindle at mitosis and to the midbody at cytokinesis. Expanded insets indicate the spindle (upper panel) and the midbody (lower panel). (B) The specificity of the staining patterns seen in A with the anti-DLG1 antibody was verified by immunofluorescence analysis of DLG1 expression in a HaCaT cell line stably transfected with an shRNA against DLG1 (left hand panel). Also shown is the DAPI staining (middle panel) for the shDLG1 cells to show the nuclei and for comparison DLG1 expression in a control cell line (right panel) stably transfected with empty vector. (C) Immunofluorescence analysis of DLG1 expression during different phases of the cell cycle following arrest with aphidicolin (G1, 0 hrs) and subsequent release through to cytokinesis (12.5 hours). (D) FACS analysis showing cell cycle progression following the aphidicolin block and release.

View larger version (47K): [in this window] [in a new window]   Fig. 2. DLG1 is phosphorylated by CDK1 and CDK2 in vitro. (A) Schematic showing the various deletion mutants used to map the regions that are phosphorylated by CDK1 and CDK2. CT, C-terminus; GUK, guanylate kinase domain; NT, N-terminus. (B) GST-DLG1 was incubated with purified CDK1 and CDK2 plus radiolabelled ATP for 20 minutes, and phosphorylation of DLG1 ascertained by SDS-PAGE and autoradiography (upper panel). The lower panel shows the Coomassie-blue-stained gels showing the control GST and the GST-DLG1 fusion protein; the arrow indicates the expected position of the non-phosphorylated GST. (C,D) GST-DLG1 and its various GST-tagged deletion mutants (Fig. 2A) were subjected to in vitro kinase assays with CDK1 and CDK2, respectively. Phosphorylation is observed in the wild type, the N-terminus and N-terminus+3 PDZ regions, but not in the C-terminal region.

View larger version (51K): [in this window] [in a new window]   Fig. 3. DLG1 mutated at Ser158 and Ser442 cannot be phosphorylated by the CDKs. (A,B) GST-tagged Ser158A, Ser442A and the double mutant in which both serines are mutated to alanine were subjected to in vitro kinase assays with CDK1 and CDK2, and analysed by SDS PAGE and autoradiography (upper panels). The lower panels show the Coomassie-blue-stained gels and confirm equal amounts of protein loading. Phosphorylation by both kinases is lost in the mutant lacking both serines.

View larger version (13K): [in this window] [in a new window]   Fig. 4. DLG1 associates in vivo with CDK1 and CDK2. Asynchronously growing HEK293 cells were extracted with E1a buffer and extracts immunoprecipitated with anti-CDK2 (A), anti-cyclin B (B) antibodies (IP) or pre-immune antibody as indicated. Co-precipitated DLG1 was detected by western blot analysis with anti-DLG1 monoclonal antibody (upper panels) and immunoprecipitated CDK2 and cyclin B detected also by western blotting (lower panels). 20% of each cell extract was used as input control. (C) HaCaT cells treated without nocodazole (–) or with nocodazole (+Noco) for 18 hours were thereafter treated with 50 µM roscovitine (+ Rosco) for 3 hours to inhibit CDK1 activity. The pattern of DLG1 expression was then analysed by western blotting with anti-DLG1 monoclonal antibody. Note the decrease in the intensity of the slower migrating forms of DLG1 in both the asynchronous and nocodazole-arrested cells following treatment with roscovitine. (D) The FACS analysis (bottom two panels) confirms M-phase arrest in nocodazole with (+) and without (–) roscovitine treatment, with the upper two panels showing asynchronously growing cells with (+) and without (–) roscovitine treatment.

View larger version (43K): [in this window] [in a new window]   Fig. 5. The phospho-point mutants migrate and localise differently. (A) HEK293 cells were transfected with the different HA-tagged DLG1 expression plasmids with a β-galactosidase expression plasmid and the pattern of DLG1 expression analysed by western blotting with anti-HA monoclonal antibody (upper panel) and anti-β-galactosidase antibody (lower panel) as a marker for transfection efficiency. (B) U2OS cells were transfected with wild-type DLG1 (top), DLG1 double Ala (Dlg1-AA) and DLG1 double Asp (Dlg1-DD) mutants and the pattern of expression analysed by immunofluorescence using anti-HA antibodies. Note the two distinct patterns of expression observed with wild-type DLG1 compared with the predominant patterns seen with the two double mutants. (C) Quantification of the percentage of cells showing the two predominant patterns of DLG1 expression (diffuse/nuclear and nuclear exclusion) from a total of 300 cells transfected in each case. (D) Immunofluorescence detection of endogenous DLG1 in HaCaT cells in the absence and presence of roscovitine (50 µM for 3 hours). Note the general decrease in the levels of DLG1 and the more diffuse cytoplasmic staining in the presence of the CDK inhibitor, although there is no change in DLG1 localisation to the midbody.

View larger version (31K): [in this window] [in a new window]   Fig. 6. The DLG1 phospho-mimic mutant has a longer half-life and is less susceptible to ubiquitylation. (A) HEK293 cells were transfected with the different HA-tagged DLG1 expression plasmids together with a β-galactosidase expression plasmid and after 24 hours the cells were treated with cycloheximide for the times indicated. Cells were then harvested and DLG1 expression ascertained by western blot analysis with the anti-HA antibody (upper panels) and anti β-galactosidase to control for transfection efficiency (lower panels). (B) The different DLG1 expression plasmids were transfected into HEK293 cells in the presence (+Ub) or absence of Flag-tagged ubiquitin expression plasmid and the levels of DLG1 expression first analysed by anti HA western blot. (C) These extracts were then subjected to immunoprecipitation with anti-HA antibody and western blot detection with anti-Flag antibody to detect the presence of ubiquitin conjugates on DLG1. Note the high degree of ubiquitylation with the wild-type DLG1 and the double Ala mutant, but very low levels of ubiquitylation on the double Asp mutant, despite readily detectable levels of DLG1 expression.

View larger version (41K): [in this window] [in a new window]   Fig. 7. DLG1 phospho-specific antibodies specifically recognise DLG1 phosphorylated by CDK1 and CDK2. (A,B) GST-DLG1 was either phosphorylated (+) or not (–) in vitro by CDK2 (A) and CDK1 (B), and then subjected to western blot analysis with anti-DLG1 monoclonal antibody (WB DLG1), the anti-Ser442-P and anti-Ser158-P antibodies. The lower panel shows the ponceau stain of the nitrocellulose membrane used for the CDK1 analysis and demonstrates equal levels of protein loading in all lanes. Note equal levels of reactivity with the anti-DLG1 antibody and specific recognition of the phosphorylated forms of DLG1 by the anti-phospho antibodies. (C) HaCaT cells were synchronised with aphidicolin and then released and cell extracts harvested at different phases of the cell cycle. DLG1 was then immunoprecipitated with anti-DLG1 monoclonal antibody, and the pattern of DLG1 expression analysed by western blotting with anti-DLG1 monoclonal antibody (WB DLG1) and the anti-Ser158-P and the anti-Ser442-P antibodies. Cell extracts were also analysed using anti-tubulin antibody to confirm equal amounts of protein extraction. Note low level of reactivity of the anti-phospho antibodies in asynchronous (Asyn) and G1-arrested cells and the specific increases in S (5 hours for Ser158, 6.5 hours for Ser442) and M phases (9 hours for Ser158) of the cell cycle. (D) Asynchronously growing HaCaT cells were untreated (–) or treated (+) with roscovitine (50 µM) for 3 hours. DLG1 was then immunoprecipitated with anti-DLG1 monoclonal antibody, and the pattern of DLG1 expression analysed by western blotting with anti-Dlg monoclonal antibody (WB Dlg) and the anti-Ser158-P antibody. Cell extracts were also analysed using anti-tubulin antibody to confirm equal amounts of protein extraction. Note the loss in reactivity of the anti-phospho antibody treatment.

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