Research Article 65 CDK phosphorylation of the discs large tumour suppressor controls its localisation and stability

Nisha Narayan, Paola Massimi and Lawrence Banks* Tumour Virology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy *Author for correspondence (e-mail: [email protected])

Accepted 16 September 2008 Journal of Cell Science 122, 65-74 Published by The Company of Biologists 2009 doi:10.1242/jcs.024554

Summary The Discs Large (Dlg) protein is known to be involved in the at the cellular junctions in G1, is enriched in the cytoplasm in regulation of cellular proliferation and polarity in a variety of S phase and locates to the mitotic spindle in M phase. We also tissues. The human homologue DLG1 is thought to be a tumour show that DLG1 is phosphorylated by both CDK1 and CDK2 suppressor, through formation of a complex with the APC on Ser158 and Ser442. These phosphorylated sites together (adenomatous polyposis coli) protein, causing negative affect the nuclear localisation of the protein, and implicate the regulation of the cell cycle. An alternative oncogenic role has role of phosphorylation on Ser158 and Ser442 in its putative also been proposed, in which the PI3-kinase pathway is activated nuclear functions as a tumour suppressor. In addition, the under the influence of the adenovirus E4 ORF1 protein. The mutants at these sites demonstrate different half-lives as well differing roles seem to be related to differences in the precise as different susceptibilities to ubiquitylation, suggesting a role pattern of expression. However, the biochemical pathways for these phosphorylation events in controlling DLG1 protein involved in regulating DLG1 function during different phases stability. These findings establish phosphorylation events as key of the cell cycle remain unclear. In this study we show that regulators of DLG1 localisation and function. phosphorylation is a major post-translational modification of the protein and it affects both location and function. DLG1 lies Key words: Dlg (Discs Large), Phosphorylation, Cell cycle

Introduction that Cdc42 and the Par6-PKCζ complex regulate the spatial The discs large homologue 1 (DLG1) protein, also known as hDlg association of DLG1 and APC, and this physical interaction or SAP-97, is a representative member of a growing family of between them is necessary for the polarisation of the microtubule proteins collectively termed membrane-associated guanylate kinase cytoskeleton (Etienne-Manneville et al., 2005). Interestingly, the

Journal of Cell Science homologues (MAGUKs), which include the ZO (zonula occludens) interaction of HPV E6, adenoviral E4 ORF1 and HTLV-1 Tax all proteins ZO1, ZO2 and ZO3 and CASK (calcium/calmodulin- interfere with the interaction between DLG1 and APC, and thereby dependent serine protein kinase). It is the mammalian homologue perturb normal cell growth control. In the case of HPV E6, this of Drosophila discs large (Dlg), and has multiple protein domains interaction is unique to those HPV types that are associated with including three PDZ motifs, an L27 domain, an SH3 domain, and the development of malignancy: so-called low risk or benign HPV a guanylate-kinase-homology (GUK) domain (Hough et al., 1997; E6 proteins do not possess the PDZ-binding motifs and do not Kim, 1997; Craven and Bredt, 1998; Gonzalez-Mariscal et al., interact with DLG1. Moreover, HPV E6 can target DLG1 for 2000). ubiquitin-mediated degradation (Gardiol et al., 1999; Pim et al., Drosophila Dlg has long been known to be involved in cell 2000; Kühne et al., 2000), probably by enhancing a normally growth control, maintenance of cell adhesion and cell polarity occurring process, because DLG1 appears to be ubiquitylated in (Bilder et al., 2000; Caruana, 2002) in both embryonic and adult cells even in the absence of E6 (Gardiol et al., 1999). The oncogenic tissues and its inactivation results in the neoplastic growth of potential of the adenovirus E4 ORF1 protein also correlates with imaginal disc epithelial cells (Bryant and Schmidt, 1990; Woods its binding to Dlg1 and other cellular PDZ proteins such as MUPP1, and Bryant, 1991; Woods and Bryant, 1993; Woods et al., 1996; MAGI1 and ZO2 via a C-terminal PDZ-binding motif. However, Goode and Perrimon, 1997). Transgenic expression of human DLG1 recent studies have shown that, upon binding to the adenovirus E4 in Drosophila harbouring Dlg mutations reverses the neoplastic ORF1, DLG1 containing the I3 isoform might also have oncogenic phenotype (Thomas et al., 1997), showing retention of the Dlg potential by activating the phosphoinositide 3-kinase (PI3K) tumour suppressor function between humans and flies. Specific PDZ pathway (Frese et al., 2006). domains of DLG1 have been shown to interact with the C-termini Numerous kinases have been shown to participate in the of several proteins, including the mitotic kinase TOPK/PBK (Gaudet phosphorylation of DLG1 and to induce functional and positional et al., 2000), the human papillomavirus (HPV) E6 oncoprotein changes in it. These include the p56lck tyrosine kinase which targets (Kiyono et al., 1997; Lee et al., 1997), the HTLV-1 Tax oncoprotein the N-terminus of DLG1 and causes coupling of tyrosine kinase (Suzuki et al., 1999), the adenoviral E4 ORF1 (Lee et al., 1997) and the voltage-gated potassium channel in T cells (Hanada et al., and the APC tumour suppressor protein (Matsumine et al., 1996). 1997), CaMKII kinase, which regulates Dlg1 localisation at the Since DLG1 forms a complex with APC, it is thought that it will synapse (Koh et al., 1999), jun N-terminal kinase (JNK), which function in processes that regulate cell polarity and proliferation in phosphorylates DLG1 in response to osmotic stress and leads to its response to cell contact in epithelial cells. A recent study has shown accumulation at cell-cell contacts (Massimi et al., 2006), the PAR- 66 Journal of Cell Science 122 (1)

1 kinase, which controls postsynaptic targeting of Dlg1 to of DLG1 expression by immunofluorescence. The results obtained neuromuscular junctions (Zhang et al., 2007) and the p38 MAPK, are shown in Fig. 1C, together with the corresponding FACS which dissociates DLG1 from GKAP and consequently triggers analysis in Fig. 1D. In the G1 population, DLG1 is highly membrane its release from the cytoskeleton (Sabio et al., 2005). bound. However its localisation progressively becomes much more Hyperphosphorylation of DLG1 has been shown to lead to its cytoplasmic as the cells progress through the cell cycle, with an interaction with the β-TrCP ubiquitin ligase receptor leading to overall diffuse staining when the cells become more rounded as ubiquitylation of the protein and a decrease in its stability (Mantovani they approach M phase. The protein shows a marked relocation to and Banks, 2003). The PDZ-binding kinase (PBK) (Gaudet et al., the mitotic spindle at mitosis and it finally accumulates strongly at 2000) is the only kinase that has so far been linked to phosphorylation the midbody at cytokinesis, in agreement with previous studies of DLG1, which regulates the cell cycle. However there is no direct (Massimi et al., 2003). Taken together, these data suggest that DLG1 evidence that DLG1 is a substrate for regulatory cell cycle kinases, is relocalised to different cellular compartments during different nor is there any information on probable biological consequences phases of the cell cycle. of its phosphorylation during the cell cycle. Here we report the phosphorylation-dependent regulation of Dlg is phosphorylated by CDK1 and CDK2 DLG1 by two principal cell cycle regulatory kinases, the cyclin- The only reported kinase potentially involved in the M phase dependent kinases 1 and 2 (CDK1 and CDK2). Both phosphorylation of DLG1 is the PDZ binding kinase (PBK) (Gaudet phosphorylation events take place on two residues within the N- et al., 2000). Since we found that DLG1 is subject to differential terminal half of the protein. Most strikingly, mutants of DLG1 that patterns of expression during the cell cycle, we hypothesised that cannot be phosphorylated or that mimic phosphorylation on those other kinases may be involved, two obvious candidates being CDK1 sites regulate the localisation and the stability of the protein. These and CDK2. To investigate whether DLG1 could be a substrate for studies directly link DLG1 function to the control by the cell cycle these kinases, we performed in vitro phosphorylation assays with and highlight the general importance of phosphorylation in the purified enzymes and purified GST-DLG1 fusion protein. As Fig. function of the protein. 2B shows, DLG1 is phosphorylated in vitro by both CDK1 and CDK2, although it is generally a better substrate for CDK2 than Results for CDK1. Cell cycle regulation of DLG1 Our next aim was to map the region(s) and possibly the residue(s) Many kinases have been implicated in the regulation of the cell that are phosphorylated by the two kinases. To do this, we used cycle, including the CDKs, the polo-like kinases (PLKs) and the constructs coding for various deletion mutants of GST-tagged Dlg mitogen-activated protein kinases (MAPKs) to name a few. They (Gardiol et al., 2002). The mutants used spanned the entire sequence function by influencing the regulation of the function of their various of the protein separately: the N-terminus, the three PDZ domains substrates (Bregman et al., 2000; Geisen and Moroy, 2002; Sherr and the C-terminal region, including the SH3 and GUK domains, and McCormick, 2002). Previous studies have also shown that as shown in Fig. 2A. The in vitro kinase assays were carried out DLG1 can act negatively to suppress G0-G1 to S phase progression with purified CDK1 and CDK2 (Fig 2C,D, respectively), which (Ishidate et al., 2000; Matsumine et al., 1996), and that it is map the major sites of phosphorylation to residues within the N-

Journal of Cell Science hyperphosphorylated during M phase (Gaudet et al., 2000). In terminus and the PDZ region of DLG1. No detectable, or at best addition, there are several reports detailing different patterns of marginal, phosphorylation of the C-terminal fragment was obtained expression of DLG1, including the presence of cytoplasmic, with CDK1 and CDK2, respectively. cytoskeletal-bound and nuclear forms of the protein (Roberts et al., The DLG1 sequence was then scanned for probable CDK 2007; Cavatorta et al., 2004), some of which are indeed regulated consensus sites (S/T-P-x-R/K, where x represents any amino acid) by different phosphorylation events (Massimi et al., 2004; Sabio et using Scansite software (http://scansite.mit.edu/), and two perfect al., 2006). However, no detailed studies have been performed to consensus sites were found containing serine residues at 158, which attempt to link these apparently diverse aspects of DLG1 localisation lies in the N terminus, and at residue 442, which lies between PDZ and function with the cell cycle. Therefore we performed a series domains 2 and 3. Upon analysis of the human, mouse, rat, of studies to investigate the pattern of DLG1 expression during the Drosophila and Caenorhabditis elegans DLG1 sequences (Table cell cycle. To do this, we first analysed the pattern of DLG1 1), it was found that though these serines and the corresponding expression in asynchronously growing HaCaT skin epithelial cells. consensus sites are perfectly conserved among the mammals and As can be seen from Fig. 1A, DLG1 expression patterns were vertebrates, they were not conserved in Drosophila and C. elegans. diverse, with cytoplasmic, diffuse and membrane-bound forms, as This suggests that these potential phosphorylation sites have roles well as structures, such as the midbody and the mitotic spindle in DLG1 function in more developed species, but that different clearly visible, suggesting potentially different patterns of pathways regulate Dlg1 in lower organisms. expression, depending upon the phase of the cell cycle. The To verify that these two potential sites are indeed phosphorylated specificity of the assay was verified using HaCaT cells stably by CDK1 and CDK2, we mutated Ser158 and Ser442 both expressing an shRNA against DLG1 (Massimi et al., 2008). As can separately and together, to alanines, to block phosphorylation on be seen from Fig. 1B these cells are devoid of detectable DLG1 the residues. The mutants expressed as GST fusion proteins were protein and the anti-DLG1 antibody used shows no signal in these then subjected to in vitro phosphorylation assays with purified cells, confirming that the different patterns of expression observed CDK1 and CDK2. The results obtained are shown in Fig 3A,B. in Fig. 1A is indeed due to specific changes in the pattern of DLG1 When either residue alone is mutated, DLG1 was still expression. To investigate this further we then proceeded to analyse phosphorylated by both kinases with almost wild-type efficiency. the pattern of DLG1 expression in HaCaT cells that had been Only when both sites were mutated was phosphorylation by CDK1 synchronised following a block with aphidicolin. Cells were and CDK2 abolished. This suggests that both serines are equally released and fixed at different times and then analysed for the pattern recognised by the CDKs as being phospho-acceptor sites. Discs large phosphorylation 67

DLG1 is found in CDK-containing complexes in vivo endogenous DLG1. Fig. 4 demonstrates that DLG1 To investigate whether DLG1 could complex with and be coimmunoprecipitates strongly with CDK2 as well as the CDK1 phosphorylated by CDKs in vivo, we first performed a series of cyclin subunit, cyclin B, although we were unable to detect an co-immunoprecipitation experiments in HEK293 cells, interaction by coimmunoprecipitation of DLG1 and CDK1. This immunoprecipitating endogenous CDK2 and then probing for might be a reflection of the relative differences in the efficiency by which CDK1 and CDK2 can phosphorylate DLG1 in vitro, with CDK2 phosphorylation of DLG1 generally being much stronger. Therefore to ascertain whether the M-phase phosphorylation of DLG1 was in part mediated by CDK1, a different approach was taken. The cells were treated for 18 hours with the microtubule- depolymerising agent nocodazole to block cells in mitosis, which was confirmed by FACS analysis (Fig. 4D). The cells were then treated with the CDK1-specific inhibitor roscovitine for 3 hours and DLG1 production was analysed by western blotting. The results obtained are shown in Fig. 4C. A number of slower migrating forms of DLG1 were present in nocodazole-arrested M-phase cells, indicating that multiple phosphorylation events take place on DLG1 in the presence of nocodazole. Intriguingly, the slowest migrating form disappears following treatment with roscovitine, demonstrating that at least one of the M-phase phosphorylation events on DLG1 is mediated by CDK1. For comparison, asynchronously growing cells were likewise treated with roscovitine and the effects on DLG1 production analysed by western blotting. As can be seen from Fig. 4C there was also a marked decrease in the intensity of the slower migrating phosphorylated form of DLG1 (Massimi et al., 2006), further supporting the notion that DLG1 is a substrate for CDKs in vivo.

Phosphorylation on Ser158 and Ser442 enhances nuclear expression of DLG1 To investigate the role of the CDK phosphorylation events on DLG1 function, we first analysed the effects of mutating these residues upon the pattern of DLG1 expression. To do this, a series of HA-tagged mutants were generated: Ser158rAla and Ser158rAsp, r r r Journal of Cell Science Ser442 Ala and Ser442 Asp, Ser158Ser442 AlaAla and Ser158Ser442rAspAsp, where mutations to alanine block phosphorylation, and those to aspartic acid mimic phosphorylation. To verify that the different DLG1 mutants were expressed, HEK293 cells were transfected with the different expression plasmids, and after 24 hours, protein levels were ascertained by western blot analysis. The results obtained are shown in Fig. 5A. Interestingly, wild-type DLG1 migrated as two distinct forms on the gel, the slower of which is more heavily phosphorylated (Caruana and Bernstein, 2001; Massimi et al., 2006), whereas the double Ala mutant co-migrated with the faster migrating band, and the double Asp mutant with the

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. 68 Journal of Cell Science 122 (1)

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 Fig. 2. DLG1 is phosphorylated by CDK1 and CDK2 in vitro. (A) Schematic lost in the mutant lacking both serines. 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 cell, and a staining that excluded the nucleus. However, the point DLG1 ascertained by SDS-PAGE and autoradiography (upper panel). The mutant Ser158Ser442rAlaAla showed a predominantly nuclear lower panel shows the Coomassie-blue-stained gels showing the control GST excluded pattern, whereas the Ser158Ser442rAspAsp had a more and the GST-DLG1 fusion protein; the arrow indicates the expected position of diffuse pattern of staining. To accurately quantify this, 300 cells were 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 counted for each assay and the expression scored as diffuse or nuclear and CDK2, respectively. Phosphorylation is observed in the wild type, the N- exclusion (Fig. 5C), which confirmed the differential pattern of terminus and N-terminus+3 PDZ regions, but not in the C-terminal region. expression of the two mutant proteins. To further demonstrate a direct link between CDK activity and the pattern of DLG1 localisation, we analysed the pattern of DLG1 expression in HaCaT cells in the presence and absence of the CDK

Journal of Cell Science slower migrating phosphorylated form of DLG1. In addition, the inhibitor roscovitine (Fig. 5D). Treatment with roscovitine induced single point mutations at Ser158 had essentially a similar phenotype, an overall decrease in the intensity of DLG1 staining and a more demonstrating that the presence or absence of a single acidic residue diffused cytoplasmic pattern of expression. However, there was no at this position has a profound effect on the overall conformational apparent change in the ability of DLG1 to localise to the midbody structure of DLG1, and that a single acidic residue at position 158 in cytokinesis, although we were unable to detect localisation to is sufficient to account for the slower migrating form of DLG1. A the mitotic spindle in the few dividing cells that could be detected. similar pattern of migration was also obtained when the mutants were These results demonstrate that inhibition of CDK activity has a direct expressed in U2OS cells (data not shown). effect upon the pattern of DLG1 expression. We then proceeded to investigate the effects of the two double point mutations upon the pattern of DLG1 expression in vivo. U2OS DLG1 protein stability and susceptibility to ubiquitylation is in cells were transfected with the different expression plasmids and part determined by its phosphorylation on Ser158 and Ser442 DLG1 expression ascertained by immunofluorescence using anti-HA The above results demonstrate that phosphorylation of DLG1 on antibodies. The results obtained in Fig. 5B show the predominant Ser158 and Ser442 can affect its cellular pattern of expression. We patterns of expression that were observed. Wild-type DLG1 exhibited also noticed from Fig. 5A subtle differences in the levels of expression two distinct patterns of expression: a diffuse staining of the entire of the different mutant forms of DLG1 and, because phosphorylation

Table 1. Comparison and sequence alignment of the regions of Dlg containing the consensus CDK phosphorylation sites in human, mouse, rat, Drosophila and C. elegans Ser158 Ser442 Homo sapiens DLG1 …FVSHSHISPIKPTEA… …GQTPASPARYSPV… Rattus norvegicus DLG1 …FVSHSHISPIKPTEA… …GQTPASPARYSPI… Mus musculus DLG1 …FVSHSHISPIKPTEA… …GQTPTSPARYSPI… Drosophila melanogaster Dlg1 …LNQRMRIESDTENA… …LSQSQSQLATSQS… Caenorhabditis elegans dlg-1 …HYLHERQRQTSHDG… …RLLIQQGTGAIFN…

Sequences were obtained from the ExPASy proteomics server (http://www.expasy.org/). Residues highlighted in red indicate the CDK consensus sequence. Discs large phosphorylation 69

of proteins has long been connected to their stability in the cell, we asked whether the phosphorylation events on these residues could affect DLG1 protein stability. Cells were transfected with the different expression plasmids, and after 24 hours, further protein synthesis was blocked by treatment with cycloheximide. The levels of DLG1 expression were then analysed over a period of 8 hours by western blotting (Fig. 6A). Strikingly, we found that the DLG1 double Ala mutant has a significantly reduced half-life compared with both wild- type DLG1 and the DLG1 double Asp mutant, with the single point mutants, not surprisingly, giving an intermediate phenotype (data not shown). This suggests that phosphorylation of the protein on these two residues increases its stability and affects the turnover of the protein. To investigate this further, we also performed an in vivo ubiquitylation assay. Cells were transfected with the HA-tagged DLG1 expression plasmids together with a Flag-tagged ubiquitin expression plasmid (Fig. 6B). The inputs for all three proteins were approximately equal; however, following immunoprecipitation of DLG1 and western blot detection for ubiquitin (Fig. 6C) we found that the wild type and the double Ala mutant show a high degree of ubiquitylation. By contrast, the double Asp mutant had only a very low level of ubiquitylation. These results suggest that the acidic status of the Ser158 and Ser442 residues can directly affect the levels to which DLG1 is ubiquitylated, which in turn affects the overall stability of the protein.

DLG1 is phosphorylated in vivo in a cell-cycle-dependent manner on Ser158 The above results provide compelling evidence that DLG1 can be a substrate for CDK phosphorylation on two different residues, and therefore suggests that phosphorylation on these sites will be cell cycle dependent. In order to verify this, we generated rabbit polyclonal phospho-specific antibodies against Ser158-P and Ser442-P. To confirm the specificity of these antibodies in western blotting, we first phosphorylated the GST-DLG1 fusion protein in

Journal of Cell Science vitro with purified CDK1 and CDK2 and cold ATP. The proteins were then subjected to gel electrophoresis and western blotting with the phospho-specific antibodies as well as with control anti-DLG1 monoclonal antibody. The anti-DLG1 monoclonal antibody equally recognised both the phosphorylated and non-phosphorylated forms of the protein (Fig 7A,B). By contrast, the phospho-specific antibodies to Ser158 and Ser442 each only recognised the CDK1 and CDK2 phosphorylated forms of GST-DLG1, confirming both the specificity of these antibodies and that the phosphorylation events actually occur on these residues. We then proceeded to investigate the pattern of reactivity of these phospho-specific antibodies on endogenous DLG1 during different phases of the cell cycle in HaCaT cells. To do this, cells were either grown asynchronously for comparison, or arrested with aphidicolin and subsequently released and analysed throughout the cell cycle. Fig. 4. DLG1 associates in vivo with CDK1 and CDK2. Asynchronously growing HEK293 cells were extracted with E1a buffer and extracts The results demonstrate that the anti-DLG1 monoclonal antibody immunoprecipitated with anti-CDK2 (A), anti-cyclin B (B) antibodies (IP) or showed a fairly constant level of reactivity against DLG1 throughout pre-immune antibody as indicated. Co-precipitated DLG1 was detected by the cell cycle (Fig. 7C) and, although there were slightly lower levels western blot analysis with anti-DLG1 monoclonal antibody (upper panels) and in early G1 phase and an apparent increase during M phase. By immunoprecipitated CDK2 and cyclin B detected also by western blotting contrast, the phospho-specific Ser158 antibodies showed a clear (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 preference for DLG1 reactivity during the S phase and the time were thereafter treated with 50 μM roscovitine (+ Rosco) for 3 hours to inhibit points leading to the M phase, compared with the much weaker CDK1 activity. The pattern of DLG1 expression was then analysed by western signals in asynchronously growing cells and in early G1. Similarly, blotting with anti-DLG1 monoclonal antibody. Note the decrease in the the phospho-specific Ser442 antibodies showed preferential intensity of the slower migrating forms of DLG1 in both the asynchronous and nocodazole-arrested cells following treatment with roscovitine. (D) The FACS reactivity against DLG1 during S phase. analysis (bottom two panels) confirms M-phase arrest in nocodazole with (+) Finally, we wanted to confirm that the Ser158 phosphorylation and without (–) roscovitine treatment, with the upper two panels showing event that we detected on DLG1 is indeed a result of CDK activity. asynchronously growing cells with (+) and without (–) roscovitine treatment. 70 Journal of Cell Science 122 (1)

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.

To do this, asynchronously growing HaCaT cells were grown in (Lahey et al., 1994; Budnik et al., 1996; Zhang et al., 2007). the absence or presence of roscovitine and DLG1 levels analysed However, the actual role of mammalian DLG1 in the control of cell using the phospho-specific antibody directed against Ser158-P. The growth and cell polarity has been a matter of much speculation.

Journal of Cell Science results obtained are shown in Fig. 7D, where it can be seen that the This is complicated by the fact that the molecular mechanisms ability of the anti-Ser158-P antibody to detect DLG1 was completely regulating DLG1 function, stability and localisation are still largely lost following treatment with roscovitine. Taken together these unknown. It has certainly been proposed to have a cell cycle results demonstrate that DLG1 is a substrate for CDKs during regulatory function (Nguyen et al., 2003) through its association different phases of the cell cycle, the consequences of which have with the APC tumour suppressor (Ishidate et al., 2000) and the profound implications for DLG1 localisation and stability. mitotic PDZ-binding kinase (Gaudet et al., 2000). Recent studies have also suggested that different isoforms under certain Discussion circumstances might also have different effects upon cell In this study, we analysed the regulation of the DLG1 tumour proliferation through activation of the PI3K pathway (Frese et al., suppressor during the cell cycle. We show that it is a substrate for 2003; Frese et al., 2006). This might in fact be linked to reported both CDK1 and CDK2 with concomitant hyper-phosphorylation differences in the cellular localisation of different DLG1 isoforms during the G1-S and M phases. Relocalisation of DLG1 was also (McLaughlin et al., 2002; Roberts et al., 2007). It now seems likely observed during different phases of the cell cycle, varying from that many of these diverse activities and patterns of expression may membrane-bound or diffuse patterns of expression, to an enhanced actually be connected through common pathways of post- degree of cytoplasmic expression during S phase, and accumulation translational modification of DLG1. The initial observations on the mitotic spindle during mitosis, as well as in the midbody supporting this came from studies which showed that DLG1 was during cytokinesis. Coupled with a mutational analysis highlighting phosphorylated via the MAPK pathway following osmotic shock, a role for acidic residues at the CDK phospho-acceptor sites on the resulting in altered patterns of protein localisation (Sabio et al., 2005; pattern of DLG1 localisation and stability, these results demonstrate Massimi et al., 2006). These studies were done under stress that CDK phosphorylation of DLG1 on Ser158 and Ser442 directly conditions, and we now show that similar alterations in the pattern contributes to its pattern of expression and stability. of DLG1 expression are obtained during a normal cell cycle and, The role of Dlg1 has been established as a regulator of cell most importantly, that this is directly controlled by both CDK1 and polarity, adhesion and junction stability (Abbott and Natzle, 1992; CDK2 by phosphorylating DLG1 on two sites, Ser158 and Ser442. Woods et al., 1996; Humbert et al., 2003), as a controller of We show that DLG1 is a substrate for CDK1 and CDK2 both in development (Caruana and Bernstein, 2001; Iizuko-kago et al., vitro and in vivo. This was done first using purified components 2007) and it has also been shown to have a strong involvement in in vitro, and mutational analysis allowed us to identify that Ser158 normalising synapse regulation at the neuromuscular junction and Ser442 were both phospho-acceptor sites for CDK1 and CDK2. Discs large phosphorylation 71

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.

The data supporting CDK regulation of DLG1 in vivo comes from and it will now be interesting to determine whether different species several experiments. We observe a strong co-immunoprecipitation of DLG1 have different levels of stability during different phases between endogenous DLG1 and CDK2, as well as between DLG1 of the cell cycle. and cyclin B, demonstrating that DLG1 can at least be found in the It is also clear that phosphorylation at Ser158 and Ser442 complexes that would be expected to have CDK activity. We also probably alters the pattern of DLG1 expression, with the double observed a clear difference in the pattern of DLG1 migration in Ala mutant exhibiting a pattern of nuclear exclusion. It is intriguing

Journal of Cell Science SDS-PAGE, when cells were exposed to the CDK inhibitor, to note that previous studies also identified Ser158 and Ser442 as roscovitine, in both G2-M as well as in asynchronously growing being sites of phosphorylation by p38γ following exposure of cells cells, suggesting that CDKs do in fact phosphorylate endogenous to osmotic stress (Sabio et al., 2005), although our studies show DLG1. We cannot formally exclude the fact that CDK5 might not that these sites are phosphorylated during the course of a normal be involved, although this seems unlikely because it is cell cycle. As a result of these phosphorylation events following predominantly expressed in neural cells. Finally, using phospho- osmotic shock, DLG1 lost its ability to bind GKAP and the specific antibodies directed against one of these sites, we show a cytoskeleton. It is tempting to speculate that phosphorylation of clear cell-cycle-dependent phosphorylation of DLG1 on Ser158 and DLG1 during a normal cell cycle by the CDKs at these two sites Ser442, with the reactivity of the antibody against Ser158 being may also have a similar function and thereby correctly regulate the lost after treatment with roscovitine. Interestingly, Ser158 appears localisation of DLG1 during different phases of the cell cycle. to be phosphorylated during late G1 and during M, whereas Ser442 Studies are now in progress to identify the cellular binding partners is predominantly phosphorylated during M phase, suggesting that of DLG1 whose interactions might be modified as a consequence a different hierarchy of phospho-acceptor site recognition on DLG1 of these phosphorylation events, with GKAP as the obvious might exist during different phases of the cell cycle. candidate (Wu et al., 2000). It is also significant to note that Ser442 These phosphorylation events appear to have a number of lies between the PDZ2 and PDZ3 domains of the protein, which is important consequences. One of them seems to be a major structural an area involved in binding several proteins involved in cell alteration. DLG1 normally migrates as two major species and propagation, including tumour suppressors such as the APC interestingly, the Ser158rAsp mutation alone is sufficient to (Matsumine et al., 1996), as well as oncoproteins such HPV E6, generate the slower migrating form of the protein, suggesting that 9ORF1 and HTLV Tax1 (Kiyono et al., 1997; Mantovani and Banks, the presence of an acidic residue at this position induces a major 2001; Latorre et al., 2005) and it will be interesting to determine structural change in DLG1. Interestingly, phosphorylation at these whether there are any cell cycle regulatory aspects to these sites also appears to regulate the level to which DLG1 is interactions. ubiquitylated. Certainly, the lack of acidic residues at Ser158 and These results provide the first direct evidence of a link between Ser442 greatly shortens the half-life of the protein, whereas by DLG1 and the regulation of the cell cycle through an interaction contrast, the presence of two acidic residues significantly extends with the key cell cycle regulators, CDK1 and CDK2, and further the half-life and decreases the level of ubiquitylation. Obviously, highlight the importance of phosphorylation as a major post- these assays were performed with ectopically expressed mutants translational modification in the life of the protein. 72 Journal of Cell Science 122 (1)

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

Journal of Cell Science 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.

Materials and Methods also cloned into the PGEX 2T plasmid to be expressed as Glutathione S-transferase Cell culture and transfections fusion proteins, by partial BamHI-EcoRI digestion of the original PCDNA3 plasmid. HaCaT, U2OS and HEK293 cells were all maintained in DMEM supplemented with 10% fetal calf serum. Cells were transfected using the standard calcium phosphate Immunoprecipitation precipitation method. The inhibitor roscovitine (Calbiochem) was used at 50 μM Total cellular extracts were prepared by lysing cells from 10 cm dishes, previously concentration and added to cells for a 3 hour incubation period pre-harvesting. washed in PBS and trypsinised, in E1A buffer [25 mM HEPES pH 7.0, 0.1% NP- 40, 150 mM NaCl, protease inhibitor cocktail I (Calbiochem)]. After incubation on Synchronisation and FACS analysis ice for 20 minutes, lysates were cleared by centrifugation at 21,000 g for 10 minutes. To synchronise HaCaT cells, aphidicolin (Sigma) was added at a concentration of 4 SDS loading buffer was then added and the extracts were analysed by SDS-PAGE μg/ml to asynchronous growing cells for 24 hours. The aphidicolin-containing medium and western blot assays. For coimmunoprecipitations, endogenous protein was was then removed and the cell culture was washed with 10 ml PBS. The PBS was immunoprecipitated from HEK293 cells extracted with E1A buffer. The soluble then replaced with complete medium. To achieve M-phase synchronisation, cells were fraction was incubated with anti-CDK2 polyclonal antibody (Santa Cruz) or anti- treated with 300 nM nocodazole for 18 hours. Cells were harvested at different times cyclin B monoclonal antibody (Calbiochem) for 3-4 hours on a rotating wheel at and DNA content was assessed by propidium iodide staining and FACS analysis as 4°C. Protein-A-Sepharose beads (GE Healthcare) were then added to the lysate and described previously (Banks et al., 1990). incubated for an additional 40 minutes at 4°C. The lysates were centrifuged and washed three times with E1A buffer and precipitated proteins were analysed by western Plasmids, primers and site-directed mutagenesis blotting. In all cases, endogenous DLG1 was detected using anti-DLG monoclonal The wild type HA-tagged DLG1 expression plasmid has been described previously antibody (2D11, Santa Cruz, Heidelberg, Germany) and HA-tagged DLG was detected (Gardiol et al., 2002). The point mutations to Ser158 and Ser442 were constructed using anti-HA monoclonal antibody (Roche, Monza, Italy). Tubulin was detected using PCR-driven site-directed mutagenesis (GeneTailor, Invitrogen). The primers using anti-tubulin monoclonal antibody (Sigma) and anti-β-galactosidase antibody for mutating Ser158 are as follows: to Ala, forward primer, 5Ј-GTCTCT - (Promega) was used to monitor transfection efficiency. Western blots were developed CACTCTCATATCGCACCCATAAAG-3Ј; and to Asp, forward primer, 5Ј-GTCTCT - using the GE Healthcare ECL System according to the manufacturer’s instructions. CACTCTCATATCGACCCCATAAAG-3Ј, reverse primer, 5Ј-GATAT GAGAG - TGAGAGACAAACCCGTGGAC-3Ј. The primers for mutating Ser442 are as GST fusion proteins and in vitro kinase assays follows: to Ala, forward primer, 5Ј-TTGGGCCAGAC TCCAG CGG CACCA - GST fusion proteins were induced and purified as described previously (Thomas et GCCAGA-3Ј; to Asp, forward primer, 5Ј-TTGGGCCA GACTCC AGCGGA - al., 1997) and their purity was tested by SDS-PAGE and Coomassie blue staining. CCCAGCCAGA-3Ј, reverse primer, 5Ј-CGCTGGAGTCT GGC CCA AGTATGA - Equal amounts of purified GST proteins were incubated with commercially purified AGACGG-3Ј. The mutations were confirmed by sequencing. The sequences were CDK1 and CDK2 kinases (New England Biolabs) for 20 minutes at 30°C in Discs large phosphorylation 73

phosphorylation buffer (20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.3 mM aprotinin, phosphatidylinositol 3-kinase by the adenovirus E4-ORF1 oncoprotein. Oncogene 22, 1 mM pepstatin) containing 56 nM [32P]ATP (Amersham) and 10 mM ATP. After 710-721. being extensively washed, the phosphorylated proteins were detected by SDS-PAGE Frese, K. K., Latorre, I. J., Chung, S. H., Caruana, G., Bernstein, A., Jones, S. N., and autoradiography. Donehower, L. A., Justice, M. J., Garner, C. C. and Javier, R. T. (2006). Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor Immunofluorescence and microscopy suppressor. EMBO J. 25, 1406-1417. Cells were fixed with 3.7% paraformaldehyde in PBS and permeabilised with 0.1% Gardiol, D., Kuhne, C., Glaunsinger, B., Lee, S. S., Javier, R. and Banks, L. (1999). Triton X-100 in PBS. Primary antibodies were incubated for 1.5 hours at 37°C, Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation. Oncogene 18, 5487-5496. followed by extensive washing in PBS and incubation for 30 minutes at 37°C with Gardiol, D., Galizzi, S. and Banks, L. (2002). Mutational analysis of the discs large secondary anti-rabbit or anti-mouse antibody conjugated with fluorescein or rhodamine tumour suppressor identifies domains responsible for human papillomavirus type 18 E6- (Molecular Probes). For visualisation of DNA, cells were stained with Hoechst 33258 mediated degradation. J. Gen. Virol. 83, 283-289. stain (Sigma). Samples were then washed several times with water and mounted with Gardiol, D., Zacchi, A., Petrera, F., Stanta, G. and Banks, L. (2006). Human discs large Vectashield mounting medium (Vector Laboratories) on glass slides. Slides were and scrib are localized at the same regions in colon mucosa and changes in their expression analysed with either a Leica DMLB fluorescence microscope equipped with a Leica patterns are correlated with loss of tissue architecture during malignant progression. Int. photo camera (A01M871016) or a Zeiss LSM 510 confocal microscope with two J. Cancer 119, 1285-1290. lasers giving excitation lines at 480 and 510 nm. The data were collected with a Gaudet, S., Branton, D. and Lue, R. A. (2000). Characterization of PDZ-binding kinase, ϫ100 or a ϫ63 objective oil-immersion lens. a mitotic kinase. Proc. Natl. Acad. Sci. USA 97, 5167-5172. Geisen, C. and Moroy, T. (2002). The oncogenic activity of cyclin E is not confined to Ubiquitylation assays Cdk2 activation alone but relies on several other, distinct functions of the protein. J. HEK293 cells were transfected with 2 μg HA-DLG1 or mutant plasmids, and 0.5 Biol. Chem. 277, 39909-39918. μg PCMV-Flag-Ubiquitin. 24 hours post transfection, the cells were treated with González-Mariscal, L., Betanzos, A. and Avila-Flores, A. (2000). MAGUK proteins: proteasome inhibitors CBZ (MG132, 50 μM, Sigma) and LLnL (50 μM, Sigma) for structure and role in the tight junction. Semin. Cell. Dev. Biol. 11, 315-324. 3 hours, after which E1A extraction was performed and the soluble fraction was Goode, S. and Perrimon, N. (1997). Inhibition of patterned cell shape change and cell incubated with anti-HA agarose beads (Sigma) to pull down DLG1 for 1-2 hours on invasion by Discs large during Drosophila oogenesis. Genes Dev. 11, 2532-2544. a rotating wheel at 4°C. The agarose beads were then extensively washed and the Hanada, T., Lin, L., Chandy, K. G., Oh, S. S. and Chishti, A. H. (1997). Human homologue of the Drosophila discs large tumour suppressor binds to p56lck tyrosine precipitated proteins were analysed by western blot using the Flag antibody (M2, kinase and Shaker type Kv1.3 potassium channel in T lymphocytes. J. Biol. Chem. 272, Sigma) to detect ubiquitin. 26899-26904. Hough, C. D., Woods, D. F., Park, S. and Bryant, P. J. (1997). Organizing a functional Half-life experiments junctional complex requires specific domains of the Drosophila MAGUK Discs large. HEK293 cells were transfected using calcium phosphate precipitation with 2 μg wild Genes Dev. 11, 3242-3253. type HA-DLG1 or the mutants, along with 0.1 μg of the β-gal plasmid as a transfection Humbert, P., Russell, S. and Richardson, H. (2003). Dlg, Scribble and Lgl in cell polarity, control. 24 hours after transfection, cells were treated for different time points as cell proliferation and cancer. BioEssays 25, 542-553. indicated with cycloheximide (50 μg/ml in DMSO) to block protein synthesis. Total Iizuka-Kogo, A., Ishidao, T., Akiyama, T. and Senda, T. (2007). Abnormal development cellular extracts were then analysed by western blot and the intensity of the bands of urogenital organs in Dlgh1-deficient mice. Development 134, 1799-1807. on the X-ray film was measured using Adobe Photoshop. Ishidate, T., Matsumine, A., Toyoshima, K. and Akiyama, T. (2000). The APC-hDlg complex negatively regulates cell cycle progression from the G0/G1 to S phase. Oncogene Phosphospecific antibodies 19, 365-372. Phospho-specific antibodies were designed against Ser158 and Ser 442 on DLG1 Kim, S. K. (1997). Polarized signaling: basolateral receptor localization in epithelial cells using the peptide sequences SHSHI(S)PIKPTE (for Ser158) and LGQTPA(S)PARYSP by PDZ-containing proteins. Curr. Opin. Cell. Biol. 9, 853-859. ., Hiraiwa, A., Fujita, M., Hayashi, Y., Akiyama, T. and Ishibashi, M. (1997). (for Ser442), and produced in rabbits by Biosense for Eurogentec, Milano, Italy. They Kiyono, T Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue were used at a dilution of 1:1000 in western blots. Appropriate secondary antibodies of the Drosophila discs large tumour suppressor protein. Proc. Natl. Acad. Sci. USA 94, conjugated to HRP were purchased from DAKO and used for western blotting at a 11612-11616. dilution of 1:2000. Koh, Y. H., Popova, E., Thomas, U., Griffith, L. C. and Budnik, V. (1999). Regulation of DLG localization at synapses by CaMKII-dependent phosphorylation. Cell 98, 353- Journal of Cell Science This work was supported by a research grant from the Associazione 363. Italiana per la Ricerca sul Cancro (AIRC). We thank members of the Lahey, T., Gorczyca, M., Jia, X. X. and Budnik, V. (1994). The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure. Neuron 13, 823- Banks lab for advice and critical comments on the manuscript. 835. Latorre, I. J., Roh, M. H., Frese, K. K., Weiss, R. 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