Proteolysis of the tumour suppressor hDlg in response to osmotic stress is mediated by caspases and independent of phosphorylation Francisco A. In ˜ esta-Vaquera 1 , Francisco Centeno 2 , Paloma del Reino 1 , Guadalupe Sabio 3 , Mark Peggie 3 and Ana Cuenda 1,3 1 Departamento de Inmunologı ´ a y Oncologı ´ a, Centro Nacional de Biotecnologı ´ a-CSIC, Madrid, Spain 2 Departamento Bioquı ´ mica y Biologı ´ a Molecular, Universidad de Extremadura, Ca ´ ceres, Spain 3 MRC Protein Phosphorylation Unit, University of Dundee, UK Mammalian cells respond to changes in the osmolarity of the medium by activating multiple signalling path- ways, with p38 mitogen-activated protein kinases (MAPKs) critical for both early response and long-term cellular adaptation to prolonged hyperosmotic exposure [1]. Although all four p38 MAPKs (p38a, p38b, p38c and p38d) are activated in response to hyperosmotic stress, activation of the isoform p38c is particularly rapid and strong compared with other p38s [2,3]. Recently, we described a novel regulatory pathway for the adaptation of cells to a hyperosmolar environment that acts parallel to the classical p38a pathway, which involves the protein kinase p38c and its substrate hDlg, and modulates the composition of the cytoskeletal protein by phosphorylating one of its components [3]. hDlg (also called Dlg1 and dlgh1) is the human orthologue of the Drosophila tumour suppressor Dlg, and belongs to the membrane-associated guanylate kinase family of scaffold proteins, whose members have a similar structural organization composed of a basic core of a variable number of PDZ domains, a SH3 domain and a catalytically inactive guanylate kinase-like region [4]. Functions of hDlg are related to the establishment and maintenance of cell polarity and the adhesion integrity of intestinal epithelial cells [5,6]. Moreover, gene-targeted mice lacking full-length hDlg show defects in the morphogenesis of the kidney and urogenital tracts [7,8]. Evidence suggests that alterations in hDlg function may contribute to the development of cancer. The Keywords apoptosis; caspase; human disc-large; osmotic shock; p38-mitogen activated protein kinase Correspondence A. Cuenda, Departamento de Inmunologı ´ ay Oncologı ´ a, Centro Nacional de Biotecnologı ´ a-CSIC, Campus de Cantoblanco-UAM, 28049-Madrid, Spain Fax: +34 91 372 0493 Tel: +34 91 585 5451 E-mail: acuenda@cnb.uam.es (Received 6 August 2008, revised 29 October 2008, accepted 7 November 2008) doi:10.1111/j.1742-4658.2008.06783.x Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38c MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating the cell with the caspase inhibitor z-VAD and is part of the apoptotic process triggered by cellular stress. Although, both caspase 3 and caspase 6 are strongly activated by osmotic shock, the time course of caspase 6 activation parallels hDlg degradation, suggesting that this caspase may be responsible for the proteolysis. Mutat- ing hDlg Asp747 to Ala abolishes caspase-induced cleavage, but does not affect the early stage of apoptosis or cell attachment. Our findings show that osmotic stress triggers hDlg degradation through a mechanism differ- ent from the one mediated by proteasomes, and we identify hDlg as a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis. Abbreviations GST, glutathione S-transferase; hDlg, human disc large; HPV, human papillomavirus; MAPK, mitogen-activated protein kinase; PSI, proteasome inhibitor I. FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 387 expression of hDlg in epithelial-derived cancers (such as cervical, gastric and colon cancers) is extremely low or even absent [9], in addition, hDlg binds to oncopro- teins expressed by viruses such as, human papillomavi- rus (HPV), human T-cell leukaemia virus type 1 and human adenovirus type 9 [10]. The binding of HPV E6 protein to hDlg causes a decrease in hDlg protein levels by inducing its proteasome-mediated degradation [11]. In epithelial cell lines, this degradation is highly dependent on the state of hDlg phosphorylation and the degree of isolation of the cell. Hyperphosphoryla- tion of hDlg makes it more susceptible to degradation induced by the HPV E6 oncoprotein [12], whereas in isolated cells, infected or not with HPV, the degrada- tion of hDlg is constitutive in the cytoplasm [13]. In addition to the suggestion that hDlg phosphory- lation may modulate its protein levels in cells, in recent years, phosphorylation has emerged as a mechanism for regulating hDlg’s function as a scaffold protein [5,14,15]. Accordingly, we have shown that hDlg is hyperphosphorylated in response to cellular stress such as osmotic shock or UV radiation. This phosphoryla- tion is mediated by p38c MAPK and triggers its dissociation from the cytoskeletal protein GKAP, therefore releasing it from the cytoskeleton into the cytoplasm [3]. Our aim in this study was to gain a better under- standing of the role of hDlg phosphorylation by p38c when cells are exposed to hyperosmotic stress. Here we analyse whether the phosphorylation of hDlg, triggered by osmotic shock, could also control levels of hDlg protein in the human epithelial cell line HeLa. We report a strong decrease in hDlg protein, although this event is independent of its phosphorylation state. More- over, this hDlg proteolysis was dependent on caspase activation during the apoptosis process in the cells. Results Osmotic shock causes a decrease of hDlg protein As mentioned previously, hDlg degradation seems to be regulated by phosphorylation and cell density [12,13,16]. Moreover, we have reported that when cells are exposed to osmotic shock, endogenous hDlg is hyperphosphorylated by the protein kinase p38c [3]. Therefore, we initiated experiments to determine whether hDlg degradation is affected by phosphory- lation mediated by p38c in a cell density-dependent manner. We first checked the regulation of hDlg phosphorylation by hyperosmotic stress at different cell densities using a phosphospecific antibody, which recognizes phosphorylated serine 158 (S158), a hDlg residue that becomes phosphorylated by p38c follow- ing sorbitol treatment [3,17]. As expected, we observed an increase in hDlg phosphorylation upon sorbitol stimulation under all experimental conditions (Fig. 1A). Moreover, a significant increase in basal hDlg phosphorylation ( 26%) was observed in con- fluent cells, but this may be due to an increase in the total amount of hDlg protein ( 30%) when cells were 100% confluent, consistent with the stabilization of this protein upon cell–cell contact (Fig. 1A) [16]. Once we had confirmed that hDlg was phosphory- lated, we examined the effect of osmotic stress on hDlg protein levels and whether this was dependent on cell density. To avoid cell death caused by long exposure to hyperosmotic shock, cells were treated for 60 min with sorbitol and then released into fresh medium for 9 or 14 h. Under these conditions, we detected a large decrease in hDlg protein levels, of 75% at 9 h and 85% at 14 h after the treatment with sorbitol ceased. Moreover, hDlg loss was similar in cells 50 or 100% confluent, indicating that cell density does not affect the decrease in hDlg protein caused by osmotic shock (Fig. 1B). We found that the levels of hDlg in HeLa cells treated with sorbitol decrease progressively with time after stress, falling to 30% at 6 h after release from sorbitol treatment (Fig. 1C). For comparison, exposure of cells to UV radiation, which also triggers hDlg phosphorylation mediated by p38c as well as its degradation [3,18], causes a decrease in hDlg protein levels similar to that observed after osmotic sock treat- ment (Fig. 1D). No strong stable accumulation of hDlg cleavage fragments could be detected at these time points (not shown), indicating that either the deg- radation of this molecule is very rapid and at multiple sites or the antibodies used do not recognize the epitope of the cleavage fragments. Osmotic shock-induced degradation of hDlg is mediated by caspase We measured the activation of different proteases after hyperosmotic shock or UV treatment in HeLa cells to determine which might be involved in hDlg degrada- tion. As shown in Fig. 2A, only caspase 3 and cas- pase 6 activities were significantly induced. Other protease activities, including caspase 8, calpain or cathepsin B, were not affected. Activation of the effec- tor caspase 3 and caspase 6 suggests that hDlg could be proteolysed by one of them. To check this, HeLa cells were treated with or without the general caspase inhibitor z-VAD prior to and during exposure to stress. As shown in Fig. 2B, z-VAD blocked hDlg degradation and the activation of caspase 3 and Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al. 388 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS caspase 6 (Fig. 2C). Other protease inhibitors, such as the calpain inhibitor MDL 28170 or the cathepsin inhibitor 3-Met adenine, did not affect hDlg degrada- tion (data not shown). Because it has been shown that the reduction in hDlg levels may be proteasome depen- dent, we also treated the cells with the proteasome inhibitor proteasome inhibitor I (PSI), and found that this did not prevent hDlg degradation (Fig. 2B), although it did prevent degradation of the kinase SGK1, the steady-state level of which is very low because of its degradation by proteasomes (Fig. 2D) [19]. These results indicate that the degradation of hDlg upon cellular stress is mediated by caspases and not by the proteasome. In addition, when we examined the time course of activation of caspases by cellular stress, we found that activation of caspase 6 is greater and more sustained than that of caspase 3 (Fig. 2E). Moreover, caspase 6 activation reached its maximum at 3 or 6 h after UV or sorbitol treatment, respectively, and then decreased. Whereas caspase 3 was transiently activated, being maximal 30–60 min after exposure to either stimulus. These results show that the loss of hDlg over the time course of sorbitol or UV treatment (Fig. 1C,D) corre- lated with the kinetics of caspase activation, particu- larly of caspase 6, supporting that hDlg cleavage is caspase dependent. Phosphorylation by p38c does not modulate hDlg proteolysis To determine whether hDlg degradation induced by osmotic stress was dependent on phosphorylation by p38c, we first expressed wild-type glutathione S-trans- ferase (GST)–hDlg, which behaves similarly to endoge- nous hDlg (Fig. S1), or different GST–hDlg mutants, in which each of the in vivo p38c-phosphorylation sites were mutated individually to Ala to prevent phosphorylation (S158A, T209A, S431A and S442A) [3]. We found that the amount of GST–hDlg wild-type and of the different GST–hDlg mutants decreased equally following osmotic shock treatment (Fig. 3A). A Sorbitol ++ hDlg (S158) hDlg (total) Cell density 50% 100% B hDlg (total) GAPDH Cell density 50% 100% Time (h) Sorbitol + 09 14 –– –– + 09 14 hDlg GAPDH 0 20 40 60 80 100 0246 hDlg protein level (%) GAPDH hDlg 0361 Time after sorbitol treatment (h) 03 6 14 Time after UV treatment (h) 9 D hDlg protein level (%) 03691215 0 20 40 60 80 100 C Time after sorbitol release (h) Time after UV treatment (h) Fig. 1. Osmotic shock causes a decrease in hDlg protein levels in a manner not depen- dent on cell density. (A) HeLa cells were exposed for 15 min to 0.5 M sorbitol. Endogenous hDlg was immunoprecipitated from 0.4 mg of cell lysate and pellets were immunoblotted using an antibody that recognizes phosphorylated hDlg [hDlg (S158)] or total hDlg (anti-hDlg). (B) Cells were exposed to hyperosmotic stress (0.5 M sorbitol) for 60 min, and then released into sorbitol-free medium for 0, 9 or 14 h. Levels of hDlg were analysed by immunoblot using hDlg Ig. (C) (Upper) HeLa cells treated as in (B). Endogenous levels of hDlg were analysed, by immunoblot with hDlg Ig, 1, 3 and 6 h after sorbitol had been washed out. (Lower) hDlg levels were quantified as described in Materials and methods. Values are means (± SE) of three independent experiments. (D) HeLa cells were exposed to UV irradiation (200 JÆm )2 ), followed by 3, 6, 9 or 14 h incubation. Endogenous levels of hDlg were analysed as in (C). Quantification values are means (± SE) of two independent experiments. Immunoblots are shown as one representa- tive experiment. Endogenous GAPDH level was used as a loading control. Lines in this figure are duplicates. F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 389 These results suggest that the phosphorylation of indi- vidual sites does not modulate hDlg degradation and prompted us to examine whether it was controlled by phosphorylation at more than one site. We then per- formed the same experiment using a hDlg mutant [GST–hDlg(A 6 )] in which all sites phosphorylated by Control Sorbitol UV-C Caspase 3 1.25 + 0.068 13.22 + 0.35 6.11 0.53 Caspase 6 1.71 + 0.26 7.74 + 1.11 4.73 + + + + 1.17 Caspase 8 1.43 + 0.03 1.67 + 0.13 1.82 0.23 Calpain 0.33 + 0.03 0.55 + 0.01 0.8 0.05 CathepsinB 0.61 + 0.08 0.47 + 0.05 + 0.36 0.02 0 3 6 9 12 0369 12 0 5 10 15 20 25 Fold caspase activation 0369 12 Time after treatment ( h ) Sorbitol UV-C Caspase 6 Sorbitol UV-C Caspase 3 E Sorbitol zVAD PSI –+ – + + – + + – – – – – – – – hDlg GAPDH –+ – – – – – – – – – – UV-C + + + + B 0 25 50 75 100 hDlg protein level (%) 0 5 10 15 Caspase activity (Fluor –1 ·min –1 ·mg protein –1 ) Sorbitol zVAD UV-C + + + ++ – –– –– –– – Caspase 3 + + + ++ Caspase 6 50 37 SGK1 PSI – + D A C Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al. 390 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS p38c in vitro and in vivo were mutated to Ala and therefore could not be phosphorylated under stress. The mutated phosphorylation sites were the four sites regulated by stresses (S158, T209, S431 and S442), one site constitutively phosphorylated in cells (S122) and one site phosphorylated in vitro by p38c (S447) [3]. Surprisingly, both hDlg wild-type and mutant disap- peared to a similar extent in cells treated with sorbitol (Fig. 3B), indicating that phosphorylation of hDlg by p38c does not regulate its degradation induced by osmotic stress in HeLa cells. To confirm these findings and verify whether hDlg degradation was dependent on its state of phosphoryla- tion, we treated cells with different MAPK pathway inhibitors to block hDlg phosphorylation [17]. We then examined hDlg loss in cells treated with inhibitors and exposed to sorbitol. Both p38MAPK inhibitors, BIRB0796, which at high concentrations inhibits all p38 and JNK isoforms [17], and SB203580, which inhibits the isoforms p38a ⁄ b, failed to abolish hDlg degradation (Fig. 3C). BIRB0796 at high concentrations (1 and 10 lm), but not SB203580, efficiently blocked hDlg phosphorylation [17] (data not shown). Because osmotic stress also may cause the activation of other MAPKs such as ERK1 ⁄ 2, ERK5 or JNK, we investigated whether these kinases were involved in hDlg degrada- tion. Treatment of cells with PD184352 at low concen- tration abolishes the activation of ERK1 ⁄ 2 and at high concentrations abolishes the activation of ERK5; treat- ment with SP600125 blocks JNK activity along with other many protein kinases such as SGK1, PRAK, AMPK, CHK, CDK2 or S6K1 [20]. None of these inhibitors blocked the decrease in hDlg protein levels (Fig. 3C) triggered by osmotic shock (or UV, data not shown), although they inhibited the different MAPKs activations (Fig. S2). These results suggest that other MAPK family members activated by cellular stress do not control the disappearance of hDlg. Fig. 2. hDlg degradation is mediated by caspases. (A) HeLa cells were treated with 0.5 M sorbitol for 60 min or with UV irradiation (200 JÆm )2 ), followed by 1 h incubation in stimulus-free media, proteases activity was determined by fluorescence emitted from specific peptide cleavage, as described in Materials and methods. Values are means (± SE) of three independent experiments. (B) Cells were prein- cubated in the absence or in the presence of 30 l M caspase inhibitor z-VAD or 60 lM proteasome inhibitor PSI, 60 min before treatment with sorbitol or UV irradiation, as in (A), followed by 14 h incubation in stimulus-free media (in the continued absence or presence of prote- ase inhibitor). Endogenous levels of hDlg were analysed and quantified as in Fig. 1C. (C) HeLa cells were preincubated with (grey bars) or without (black bars) the pan-caspase inhibitor z-VAD for 1 h before treatment with sorbitol or UV irradiation, as in (A). Values are means (± SE) of three independent experiments. (D) HeLa cells were incubated with or without 60 l M PSI and endogenous levels of SGK1 were analysed by western blotting using SGK1 Ig. (E) HeLa cells (circles) or HEK293 cells (triangles) were exposed, as in (A), to sorbitol (black circles or triangles) or to UV irradiation (empty circles or triangles), then released from the stimuli for the times indicated before assaying caspase activities. Values are means (± SE) of two independent experiments. C Sorbitol BIRB0796 (µM) SB203580 (µM) PD184352 (µM) SP600125 (µM) +++ + – ++ + + – ++ – – – – – – 10 – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.1 1 10 – 10 – – 2 – –– – 10 – – – – – – hDlg GAPDH Fig. 3. The decrease in hDlg protein level is not regulated by p38c phosphorylation. (A) HeLa cells were transfected with either GST–hDlg wild-type or different GST–hDlg mutants in which in vivo p38c phosphorylation sites had been mutated to Ala, and then exposed 0.5 M sorbi- tol for 60 min. Overexpressed GST–hDlg were analysed 6 h after sorbitol had been washed out, as in Fig. 1. (B) Cells were transfected with GST–hDlg wild-type or GST–hDlg(A 6 ) mutant. Cells were treated as in (A) and GST–hDlg was analysed 1, 3 and 6 h after sorbitol release. Lines indicate duplicates. (C) Cells were preincubated for 2 h with or without different kinase inhibitors, at the concentrations indicated. Cells were exposed to 0.5 M sorbitol for 60 min, and then incubated for 9 h in sorbitol-free medium (in the continued absence or presence of inhibitor). Endogenous levels of hDlg were analysed as in Fig. 1C. GAPDH or p38a levels were used as loading controls. F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 391 To determine whether hDlg phosphorylation regu- lates hDlg degradation and to exclude the possibility that other phosphorylation sites, different from those for p38c, may modulate hDlg degradation, we treated cells with the general kinase inhibitor staurosporine [21]. To test whether staurosporine could inhibit hDlg phosphorylation, cells were preincubated with or with- out this compound for 1 h before exposure to sorbitol. Phosphorylation of endogenous hDlg was blocked completely by 1 lm staurosporine (Fig. 4A). However, preincubation of cells with staurosporine failed to block the decrease in hDlg protein level, and caused a significant increase in hDlg degradation (Fig. 4B). Given the above results, we decided to check whether incubation of cells with staurosporine alone caused hDlg loss. As shown in Fig. 4C, staurosporine also caused hDlg degradation in a concentration- and time- dependent manner. hDlg is degraded in apoptotic cells These findings suggest that hDlg degradation might be related to the apoptosis of the cell, because stauro- sporine is a potent inducer of caspase-dependent cell apoptosis [21] (data not shown). Therefore, we estab- lished that, after cellular stress, cells undergo apopto- sis. HeLa, or HEK293 cells for comparison, were exposed to either sorbitol or UV, and apoptosis was determined 3 or 14 h after exposure to the stimulus. As shown in Fig. 5A,  70% of HeLa cells underwent apoptosis 14 h post stimulus, whereas only  15% of HEK293 cells started to die 14 h after sorbitol or UV treatment. Basal levels of apoptosis in control cells, not exposed to stress, were also significantly higher in HeLa cells than in HEK293 cells (Fig. 5A). Accord- ingly, when we examined the levels of hDlg protein in HEK293 cells that had been exposed to sorbitol or UV, we could not detect any protein degradation even 14 h after the stimulus (Fig. 5B). However, neither 0 25 50 75 100 hDl gprotein level (%) Staurosporine (µM) 2 0110 1 10 GAPDH hDlg 0 Staurosporine (µM) 7 2 727Time (h) C B 0 25 50 75 100 hDlg protein level (%) Staurosporine (µM) Sorbitol 00 1 10 – ++ + GAPDH hDlg Staurosporine (µM) Sorbitol 0 01 –+ + hDlg (S158) hDlg (total) A hDlg (T209) hDlg (S431) hDlg (S442) Fig. 4. The broad-spectrum kinase inhibitor staurosporine enhances hDlg degradation. (A) Cells were incubated for 1 h with or without the indicated concentration of staurosporine and then exposed for 15 min to 0.5 M sorbitol. hDlg was immunoprecipitated and analy- sed using antibodies that recognize phosphorylated hDlg at four different residues, hDlg (S158), hDlg (T209), hDlg (S431) or hDlg (S442), or an antibody that recognizes total hDlg (anti-hDlg). (B) HeLa cells were preincubated for 1 h with or without the pan- kinase inhibitor staurosporine at the concentrations indicated, and treated with 0.5 M sorbitol for 60 min, and then incubated for 3 h in sorbitol-free medium (in the continued absence or presence of the inhibitor). Endogenous levels of hDlg were analysed by immuno- blotting (lower) and the percentage of protein level quantified (upper) as described in Materials and methods. Values are means (± SE) of three independent experiments. (C) HeLa cells were trea- ted without or with 1 or 10 l M staurosporine for 2 h (upper and lower) or 7 h (lower). Endogenous levels of hDlg were analysed as described previously and the percentage of hDlg protein was quantified (lower). Values are means (± SE) of two independent experiments. Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al. 392 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS caspase 3 nor caspase 6 were activated in HEK293 cells under any of the experimental conditions described (Fig. 2E), which is consistent with the lack of hDlg degradation observed in this cell line. The low percentage of HEK293 cells undergoing apoptosis (compared with HeLa cells), and the the lack of hDlg loss in HEK293 cells, support the idea that hDlg deg- radation in HeLa cells is part of the apoptotic event triggered by cellular stress. Changes in hDlg localization upon hyperosmotic shock exposure Although hDlg is normally localized in adherens junc- tions at sites of cell–cell contact, its cellular distribu- tion is different in confluent and subconfluent cells, hence our next question was whether this localization would change during apoptosis induced by sorbitol. As expected, when HeLa cells were 50% confluent, hDlg was localized diffusely throughout the cytosol and at the membrane. hDlg localization changed when cells reached confluency; in this condition, hDlg was present mainly at the membrane, whereas the amount found in the cytoplasm decreased markedly (Fig. 6A). These results were confirmed by subcellular fractionation analysis; we found that the amount of hDlg in the cytoplasm is greater in subconfluent cells, but in confluent cells hDlg is mainly in the membrane frac- tion (Fig. 6B). After exposing the cells to sorbitol (Fig. 6A,B), the total amount of hDlg decreased in both 50% and 100% confluent cells, and this decrease was equal in all cell compartments (Fig. 6B). These results show that hDlg localizes to the plasma mem- brane when cells reach confluency and establish cell– cell contact, and that its degradation occurs in all cell compartments in which hDlg is present. In addition, in CaCo-2 cells, derived from human colonic adenocarcinoma and in which hDlg is degraded in response to osmotic stress (Fig. S3), hDlg was found mostly in areas of cell–cell contact and a substantial and gradual loss from the mem- brane was observed after osmotic shock treatment (Fig. 6C). The more compact localization of hDlg is partially lost and hDlg is localized more diffusely throughout the cytoplasm in the vicinity of the mem- brane (Fig. 6Ca–c). However, pretreatment of the cell with the p38MAPK inhibitor BIRB0796 causes a sig- nificant delay in the loss of Dlg from the membrane (Fig. 6Cd–f), whereas pretreatment with the caspase inhibitor z-VAD, in both the absence and presence of BIRB0796, preserved the membrane localization of hDlg under all experimental conditions (Fig. 6Cg– l). Because cell–cell contacts are largely preserved at these initial time points, it is suggested that hDlg Sorbitol 0 20 40 60 80 03 14 UV-C 03 14 Time after stimuli treatment (h) Apoptosis(%) A B Time after stimuli treatment (h) C0 3 6 14 Sorbitol UV-C hDlg GAPDH hDlg GAPDH Fig. 5. Cellular stresses induce apoptosis in HeLa cells. (A) HeLa (black bars) or HEK293 cells (grey bars) were exposed to 0.5 M sorbitol for 60 min or to UV irradiation (200 JÆm )2 ) followed by 3 or 14 h incubation in stimulus-free media before quantitative analysis of apoptosis by propidium iodide ⁄ annexin V. Values are means (± SE) of three independent experiments. (B) HEK293 cells were exposed to 0.5 M sorbitol for 60 min or to UV irradiation (200 JÆm )2 ), followed by 0, 3, 6 or 14 h incubation. Endogenous levels of hDlg were analysed as described in Fig. 1. F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 393 A Control Sorbitol Cell density 50% 100% Control Sorbitol hDlg hDlg + DAPI hDlg hDlg + DAPI hDlg hDlg + DAPI hDlg hDlg + DAPI hDlg InsR Calpain Cell density 50% 100% Cytop CytopCytopCytopMemb MembMembMemb Control Sorbitol Control Sorbitol B No inhibitor zVAD Treatment None 1 h Sorbitol 1 h Sorbitol + 3 h release BIRB0796 + zVAD hDlg hDlg hDlg hDlg C a b c d e f g h i j k l BIRB0796 Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al. 394 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS cleavage represents a step in apoptosis that may precede cell–cell detachment. Identification of hDlg caspase-cleavage sites These findings suggest that hDlg is a caspase target. To confirm this and identify the caspase(s) cleavage site(s) on hDlg, experiments were performed using mutations that affect putative caspase 3 and ⁄ or cas- pase 6 sites. Within their substrates, caspases recog- nize a core tetrapeptide motif (P 4 P 3 P 2 P 1 ) that contains an essential aspartic acid residue required for the cleavage reaction at position P 1 [22]. One well-defined substrate-recognition motif for caspases, in particular caspase 3, is DXXD, whereas for cas- pase 6 it is (I ⁄ V ⁄ L)EXD [22]. Analysis of the hDlg protein sequence indicates that it contains several putative caspase-cleavage sites, DVRD 255 and DGRD 750 , which are typical caspase 3 recognition sequences, YEVD 747 , which may be another potential caspase site, and QSVD 397 , which has been previ- ously reported as an unusual caspase 3 cleavage site [18]. We generated different hDlg mutants in which the aspartic acid residue predicted as being required for caspase cleavage was mutated to Ala (Fig. 7A). These constructs were transfected to HeLa cells and the effects of sorbitol (and UV) treatment on GST– hDlg wild-type or mutant GST–hDlg degradation were compared. As shown in Fig. 7B, 3 h after stim- ulation ceased, only the mutation D747A blocked the degradation of GST–hDlg, although wild-type hDlg and the other hDlg mutant forms were degraded to the same extent after treatment. Quanti- fication of hDlg protein confirmed that D747A was the only mutant not cleaved after cellular stress (Fig. 7C). These results identify the sequence YEVD as a possible site of hDlg cleavage in early apoptotic cells. Effect of hDlg cleavage on cell–cell detachment and early stage of apoptosis To evaluate the role of hDlg during these processes, HeLa cells were transfected with hDlg wild-type or mutant hDlgD747A, which is not degraded, or mutant hDlgD397A, as a control. We tried to generate cell lines stably overexpressing wild-type or hDlg mutants. However, none of the attempts was successful. There- fore, the transfection procedure was optimized to Control Sorb. UV-C Tubulin Tubulin Tubulin Tubulin Tubulin hDlg(WT) hDlg(D255A) hDlg(D397A) hDlg(D255A/D750A) hDlg(D747A) B C 0 20 40 60 80 100 hDl g -WT -D255A -D397A -D747A -D255A/D750A hDlg protein level (%) A PDZ1 PDZ3 SH3 GUKPDZ2 hDlg DVRD 255 QSVD 397 YEVD 747 DGRD 750 Fig. 7. Identification of the hDlg-caspase-cleavage site. (A) Sche- matic representation of hDlg and the putative caspase-cleavage sites. (B) Cells were transfected with GST–hDlg wild-type or differ- ent GST–hDlg mutants, in which different caspase-cleavage sites have been mutated to Ala, and then exposed to 0.5 M sorbitol for 60 min or UV irradiation (200 JÆm )2 ). GST–hDlg were analysed 3 h after UV treatment or after sorbitol had been washed out, as in Fig. 1. The endogenous tubulin level was use as the loading con- trol. Immunoblots are shown as one representative experiment. (C) Percentage GST–hDlg protein level was quantified as before: hDlg protein from control cells (black bars), cells treated with sorbitol (dark grey bars) or treated with UV (light grey bars). Values are means (± SE) of three to four independent experiments. Fig. 6. Localization of hDlg in apoptotic cells. (A) HeLa cells were grown at 50 or 100% confluency, exposed or not to 0.5 M sorbitol for 60 min, stained with hDlg Ig 3 h after sorbitol release, and subjected to immunofluorescence microscopy. Nuclei are stained with DAPI. Similar results were obtained in three independent experiments. (B) HeLa cells were exposed to 0.5 M sorbitol for 60 min, and then released in sorbitol-free medium for 0 or 9 h. Cells were subjected to cellular fractionation as indicated in Materials and methods and 10–30 lgof protein from cytoplasm and membrane fractions were immunoblotted using the antibodies indicated. (C) CaCo-2 cells were preincubated with or without the kinase inhibitor BIRB0796 (1 l M) and in the presence or absence of z-VAD (30 lM). Cells were exposed to hyperosmotic stress for 1 h followed by 0 or 3 h incubation in sorbitol-free medium. F. A. In˜ esta-Vaquera et al. Caspase-dependent degradation of hDlg FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS 395 result in  50% hDlg-transfected HeLa cells, which is a level tolerated by the cells. Using these cells, we examined the effect of hDlg mutation on the progres- sion of both cell detachment and apoptosis induced by sorbitol. As shown in Fig. 8, none of these processes was significantly affected by the mutation D747A of hDlg, indicating that its proteolysis is not sufficient for the regulation of early apoptotic events. Discussion Our aim was to gain a better understanding of the role of hDlg phosphorylation in the adaptation of cells to stress, by determining whether stress could regulate hDlg degradation in cells exposed to changes in envi- ronmental osmolarity. We analysed the extent of hDlg phosphorylation and hDlg protein levels in subconflu- ent and confluent cells exposed to hyperosmolarity. Our results show that hDlg is phosphorylated in response to osmotic stress, although the degree of hDlg phosphorylation upon sorbitol treatment is  40% higher in subconfluent cells than in confluent cells. This is probably because of the difference in hDlg localization observed. In confluent cells, hDlg is present mainly in the membrane, at sites of cell–cell contact. However, in subconfluent cells, hDlg localiza- tion is identical to its physiological kinase p38c, and both are localized diffusely throughout the cytosol, nucleus and membrane [3]. Under these conditions, hDlg may be more accessible to the kinase and this may facilitate its phosphorylation after sorbitol treat- ment. In addition, we have also shown that hDlg is largely degraded in cells exposed to hypertonicity and that this is not dependent on cell density. As mentioned previously, our objective was to inves- tigate in more depth the role of phosphorylation in hDlg degradation, but the results obtained from exper- iments using hDlg mutated at different p38c phosphor- ylation sites or using several kinase inhibitors, including the general kinase inhibitor staurosporine, demonstrated that under these conditions hDlg degra- dation was not regulated by its phosphorylation state. In addition, we showed that hDlg degradation is blocked by z-VAD, a general caspase inhibitor, and this indicates that hyperosmotic shock-induced loss of hDlg is mediated by caspases during apoptosis. The caspase-dependent cleavage of many key structural and regulatory proteins contributes to the typical morphological changes, including the dismantling of cell–cell contact, seen during apoptosis. As mentioned previously, hDlg is a scaffold protein, which has been implicated in the maintenance of cell polarity and cell adhesion [4]. We report that hDlg is proteolysed by caspases during the apoptosis of HeLa (Fig. S3) trig- gered by hyperosmolarity and also in CaCo-2 cells and mouse embryonic fibroblasts. However, the mechanism by which there is a different degree of apoptosis, and therefore of hDlg degradation, in HeLa cells than in HEK293 cells is unknown. We speculate that in HeLa cells prolonged exposure to hypertonicity induces apoptosis because of a lack of or the dysfunction of the component(s) needed for the adaptive response of these cells to stress. For example, it has been described that, in some cell types, the lack of the restoration of cell volume after cell shrinkage is associated with the concomitant appearance of apoptosis [23]. However, the difference in the degree of apoptosis observed between HeLa and HEK293 cells may be due to a dif- ference in the sensitivity of these cells to the strength 0 5 10 15 SorbitolControl Caspase 3 activity (Fluor –1 ·min –1 ·mg protein –1 ) hDlg–D747A hDlg–D397A hDlg–WT None B 3 4 5 6 7 06 Time after sorbitol release (h) hDlg–D747A hDlg–D397A hDlg–WT None Number of attached cell (x10 –5 ) A Fig. 8. Quantification of cells attached and apoptotic cells after sor- bitol treatment. (A) Cell attachment and (B) caspase 3 activation were measured as indicated in Materials and methods, at the times indicated – (A) 0 and 6 h or (B) 0 and 2 h – in nontransfected and hDlg wild-type or hDlg–D397A or hDlg–D747A transfected HeLa cells. All other experimental details are described in the text. Values are means (± SE) of three independent experiments performed in triplicate. Caspase-dependent degradation of hDlg F. A. In˜ esta-Vaquera et al. 396 FEBS Journal 276 (2009) 387–400 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... kinase domain, the region by which hDlg binds to proteins such as GKAP, which target it to the cytoskeleton [3]; cleavage at this site would release hDlg from this cellular compartment These findings indicate that when apoptosis is initiated in HeLa cells exposed to hypertonicity, activation of caspases causes the proteolysis of hDlg at YEVD747 and this releases it from the cortical cytoskeleton at the. .. that hDlg is associated with the submembranous cytoskeleton at cell–cell contact via an E-cadherin-induced assembly of the cortical cytoskeleton [27], we suggest that the change in hDlg localization may be caused by the possible loss of E-cadherin or other protein(s) in the hDlg complex under stressful conditions, and the cortical cytoskeleton is therefore disrupted and hDlg released into the cytoplasm... localization because in cells pretreated with the p38MAPKs inhibitor, hDlg is not phosphorylated [17] and a significant delay in its release from the vicinity of the membrane to the cytoskeleton is observed These results are in agreement with previous findings in which phosphorylation of hDlg, catalysed by p38c, triggers its dissociation from the scaffold protein GKAP and its release from the cytoskeleton [3] However,... disruption of the scaffolding ability of hDlg by caspase cleavage may be one of the major steps in apoptosis, which is the dismantling of the cell– cell contacts, therefore causing cell–cell detachment under osmotic stress In conclusion, our studies demonstrate that hDlg is proteolysed in vivo by caspases via a different mechanism to that described previously, which was proteasome- and phosphorylation- dependent... membrane into the cytoplasm where hDlg may be further proteolysed In immunolocalization experiments we observed that, after osmotic shock treatment, the more compact localization of hDlg at the cell–cell contact region is lost and it is more diffusely localized throughout the cytoplasm at the vicinity of the membrane We cannot exclude the possibility that hDlg phosphorylation may contribute to its change in. .. E9 Supporting information The following supplementary material is available: Fig S1 Exogenous GST hDlg degradation is mediated by caspases Fig S2 Blockade of different MAPK pathway activation by different kinase inhibitors Fig S3 Osmotic shock causes a decrease in hDlg levels in CaCo-2 cells and mouse embryonic fibriblasts This supplementary material can be found in the online version of this article... 505 nm The substrate used for calpain activity was N-succinylLeu-Leu-Val-Tyr-7-amino-4-methylcoumarin (kexc 380 nm and kem 440 nm) and the measurements were carried out in the presence of 100 lm Ca2+ in the reaction buffer The substrate used for assaying cathepsin B activity was Z-Arg-Arg-7-amino-4-methylcoumarin (kexc 380 nm and kem 440 nm) and the measurements were carried out in the presence of 0.6... anti-calpain for the cytoplasmic fraction and anti-(insulin receptor) for the membrane fraction Propidium iodide/annexin V assay HeLa or HEK293 cells were exposed to UV radiation (200 JÆm)2) or to hyperosmotic shock (0.5 m sorbitol) for 1 h and then released into fresh media for 3 or 14 h before counting apoptotic cells using an annexin V ⁄ FITC detection kit (Oncogene, Inc Boston, MA, USA) according to manufacturer’s... cytoplasm The lack of effect of an hDlg mutant, which cannot be proteolysed at initial apoptosis, on cell detachment or early apoptosis suggests that hDlg or its cleavage fragments do not have an active role in apoptotic progression However, at later times, this mutant is also degraded and, given the importance of the hDlg protein level in several processes, we cannot discount the possibility that disruption... (Division of Signal Transduction Therapy, University of Dundee), coordinated by Dr H McLauchlan and J Hastie, for generation and purification of antibodies FAI-V was supported by fellowship from the Spanish Government (Beca FPU, Ministerio de Educacion y Ciencia) The work in the author’s laboratory is supported by the Medical Research Council UK, pharmaceutical companies that support the Division of . Proteolysis of the tumour suppressor hDlg in response to osmotic stress is mediated by caspases and independent of phosphorylation Francisco A. In ˜ esta-Vaquera 1 ,. protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating