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• W2007352125 abstract "Article24 February 2005free access p38γ regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP Guadalupe Sabio Guadalupe Sabio MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Departmento Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain Search for more papers by this author James Simon Campbell Arthur James Simon Campbell Arthur MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Yvonne Kuma Yvonne Kuma MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Mark Peggie Mark Peggie MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Julia Carr Julia Carr MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Vicky Murray-Tait Vicky Murray-Tait School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Francisco Centeno Francisco Centeno Departmento Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain Search for more papers by this author Michel Goedert Michel Goedert MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK Search for more papers by this author Nicholas A Morrice Nicholas A Morrice MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Ana Cuenda Corresponding Author Ana Cuenda MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Guadalupe Sabio Guadalupe Sabio MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Departmento Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain Search for more papers by this author James Simon Campbell Arthur James Simon Campbell Arthur MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Yvonne Kuma Yvonne Kuma MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Mark Peggie Mark Peggie MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Julia Carr Julia Carr MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Vicky Murray-Tait Vicky Murray-Tait School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Francisco Centeno Francisco Centeno Departmento Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain Search for more papers by this author Michel Goedert Michel Goedert MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK Search for more papers by this author Nicholas A Morrice Nicholas A Morrice MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Ana Cuenda Corresponding Author Ana Cuenda MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Author Information Guadalupe Sabio1,3, James Simon Campbell Arthur1, Yvonne Kuma1, Mark Peggie1, Julia Carr1, Vicky Murray-Tait2, Francisco Centeno3, Michel Goedert4, Nicholas A Morrice1 and Ana Cuenda 1 1MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK 2School of Life Sciences, University of Dundee, Dundee, UK 3Departmento Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain 4MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK *Corresponding author. MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK. Tel.: +44 1382 344241; Fax: +44 1382 223778; E-mail: [email protected] The EMBO Journal (2005)24:1134-1145https://doi.org/10.1038/sj.emboj.7600578 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38γ MAP kinase (SAPK3/p38γ) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38γ-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular–junctional complexes, and cell shape and volume in response to osmotic stress. Introduction Cells respond to changes in the physical and chemical properties of the environment by altering many cellular functions. These environmental changes include alterations in the concentrations of nutrients, growth factors, cytokines and cell-damaging agents, but also physical stimulation mediated by changes in the osmolarity in the medium. When exposed to hyperosmotic stress, eukaryotic cells shrink due to the efflux of water from the cell (Morris et al, 2003). For cellular integrity and homeostasis to be maintained under these conditions, cells employ adaptive responses to restore cell volume (O'Neill, 1999) and reinforce the cytoskeletal architecture (Di Ciano et al, 2002). Mammalian cells respond to changes in the osmolarity by activating signalling pathways that involve the activation of p38 mitogen-activated protein kinases (MAPKs) (Kyriakis and Avruch, 2001) which are critical for long-term cellular adaptation to prolonged hyperosmotic exposure. The changes triggered by this pathway include alterations in gene transcription (Sheikh-Hamad et al, 1998; Garmyn et al, 2001) and post-translational modification of cytoskeletal remodelling proteins (Landry and Huot, 1999; Bustamante et al, 2003). Although all four p38 MAPKs (p38α, p38β, SAPK3/p38γ and SAPK4/p38δ) are activated in mammalian cells in response to hyperosmotic stress, the activation of SAPK3/p38γ is particularly rapid and strong compared to other p38s (Goedert et al, 1997a; Sabio et al, 2004). Here we identify a novel physiological substrate for SAPK3/p38γ, which is likely to play an important role in the adaptive response to osmotic stress. To identify new substrates of SAPK3/p38γ, we have exploited a characteristic of this kinase that is unique among all MAPK family members, namely the presence of a carboxyl-terminal sequence which can dock with the PDZ domains of different proteins. Moreover, the phosphorylation of PDZ-domain-containing proteins by SAPK3/p38γ is dependent on this interaction (Hasegawa et al, 1999; Sabio et al, 2004), suggesting that such proteins are likely to be its physiological substrates. One of these proteins is the synapse-associated protein 97 (SAP97/hDlg), the mammalian homologue of the Drosophila tumour suppressor gene dlg, which is a member of a family of membrane-associated guanylate kinase (GK) homologues (MAGUKs) (Garner et al, 2000). SAP97/hDlg is found at the pre- and post-synaptic density in neuronal cells and at the region of cell–cell contacts along the epithelial lateral membrane. SAP97/hDlg has also been localised at the neuromuscular junction in muscle and at the lymphocyte immune synapse (Muller et al, 1995; Reuver and Garner, 1998; Leonoudakis et al, 2001). Structurally, SAP97/hDlg is composed of three N-terminal PDZ domains, followed by an Src homology 3 (SH3) domain and a GK-like region (Garner et al, 2000). Functionally, SAP97/hDlg is a scaffolding protein that assembles multi-component protein complexes; thereby facilitating signal transduction, and it has also been implicated in maintaining cell adhesion and cell polarity (Caruana, 2002; Humbert et al, 2003). An important unresolved issue, with regard to the ability of SAP97/hDlg to build multi-component protein complexes, is which kinases and phosphatases act directly on it to regulate its association with different binding partners. In this study, we have found that SAPK3/p38γ binds directly to two PDZ domains of SAP97/hDlg and phosphorylates it in vivo in response to cellular stress. Moreover, we show that the phosphorylation of SAP97/hDlg by SAPK3/p38γ regulates its association to the GK-associated protein (GKAP) and its localisation in the cytoskeleton. Results Phosphorylation of SAP97/hDlg by SAPK3/p38γ depends on their interaction Immunolocalisation studies showed that in HeLa, PC12 and SH5-SY5Y cells SAPK3/p38γ and SAP97/hDlg both had the same diffuse cytoplasmic and nuclear localisation (Figure 1A), and in PC12 along neuritic processes (Figure 1A VII–IX), indicating a possible interaction between them in these cellular compartments. This was confirmed by the finding that SAPK3/p38γ co-immunoprecipitated with SAP97/hDlg (Figure 1B). Since SAP97/hDlg contains three PDZ domains, we first assessed the importance of the C-terminus of SAPK3/p38γ on the binding to SAP97/hDlg. We therefore examined whether these proteins co-immunoprecipitated from extracts of human embryonic kidney (HEK)293 cells transfected with either SAPK3(FL) (full length) or SAPK3(ΔC) (lacking the last four amino acids). As predicted, endogenous SAP97/hDlg only co-immunoprecipitated with FL SAPK3/p38γ (Figure 1B). These results indicate that the last four amino acids of SAPK3/p38γ are essential for its association with the PDZ domain of SAP97/hDlg. Secondly, to determine which SAP97-PDZ domain was responsible for the interaction, cells were transiently transfected with DNA constructs encoding the different SAP97-PDZ domains together with SAPK3/p38γ. We found that SAPK3/p38γ interacted with PDZ domains 1 and 3 of SAP97/hDlg in pulldown and co-immunoprecipitation experiments (Figure 1C). Figure 1.Phosphorylation of SAP97/hDlg by SAPK3/p38γ depends on the interaction of these two proteins. (A) HeLa cells (I, II, III), differentiated PC12 (IV–IX) and differentiated SH5-SY5Y neuroblastoma (X, XI, XII) were stained with anti-SAPK3 or anti-SAP97 antibody, and subjected to fluorescence microscopy. SAPK3/p38γ and SAP97/hDlg are shown in green and red, respectively. In merged images, the co-localised signal is shown in yellow. Scale bar, 10 μM. (B) Co-immunoprecipitation of SAPK3/p38γ with SAP97. HEK293 cells were transfected with GFP-SAPK3(FL) or GFP-SAPK3 (lacking the last four amino acids (ΔC)). Endogenous SAP97 in the lysates was immunoprecipitated, and immunoblotted with anti-SAPK3 or anti-SAP97 antibodies. GFP-SAPK3 was immunoprecipitated using an anti-GFP antibody, and immunoprecipitates immunoblotted with anti-SAP97 or anti-SAPK3 antibodies. (C) Interaction of SAPK3/p38γ with SAP97PDZ domains. HEK293 cells were transfected with GFP-SAPK3(FL) and either GST, GST-SAP97(PDZ1), GST-SAP97(PDZ2) or GST-SAP97(PDZ3). After transfection the cells were lysed and GST-fusion proteins purified by affinity chromatography on GSH-Sepharose beads. The GFP-SAPK3 was immunoprecipitated using an anti-GFP antibody. The proteins were immunoblotted using an anti-SAPK3/p38γ antibody to detect GFP-SAPK3/p38γ, or anti-GST antibody to detect expression of GST-SAP97 fusion proteins. (D) Phosphorylation of SAP97 by SAPK3/p38γ is dependent on the carboxy-terminal four amino acids of the kinase. GST-SAP97 or MBP, both at 1 μM, were phosphorylated for the times indicated with 2.0 U/ml of either GST-SAPK3(FL) or GST-SAPK3(ΔC). The results are shown as the mean±s.e.m. of four experiments. (E) GST-SAP97 (filled bars) or MBP (open bars), each at 1 μM, were incubated for 30 min at room temperature with synthetic peptides (300 μM) corresponding to the C-terminal six (PKETAL) or eight (RVPKETAL) amino acids of rat SAPK3. GST-SAPK3/p38γ (black bars) or GST-SAPK4/p38δ (grey bars) were added to 0.2 U/ml and the reactions initiated with Mg[γ-32P]ATP. Substrate phosphorylation is plotted as a percentage of that measured in the absence of each peptide. Results in (E) are shown as the mean±s.e.m. for triplicate determinations from a single experiment. Download figure Download PowerPoint Moreover, the phosphorylation of SAP97/hDlg by SAPK3/p38γ in vitro was dependent on the extreme C-terminus of SAPK3/p38γ. SAPK3(ΔC) phosphorylated SAP97/hDlg very poorly, though it phosphorylated myelin basic protein (MBP; a protein devoid of PDZ domains) as well as full-length SAPK3(FL) (Figure 1D). Also, pre-incubation of SAP97/hDlg with synthetic peptides corresponding to the C-terminal six or eight amino acids of SAPK3/p38γ prevented phosphorylation of SAP97/hDlg by SAPK3/p38γ, but not by SAPK4/p38δ (Figure 1E). Phosphorylation of SAP97/hDlg after cellular stresses We investigated whether other members of the MAPK family could phosphorylate SAP97/hDlg and SAP102, another PDZ-domain-containing member of the MAGUK family. We compared initial rates of phosphorylation by different MAPK family members in vitro and showed that SAP97/hDlg was phosphorylated efficiently by SAPK3/p38γ or SAPK4/p38δ, whereas p38α, p38β, c-jun N-terminal kinase (JNK)1-3 or extracellular signal regulated kinase-2 (ERK2) only phosphorylated SAP97/hDlg much more slowly. In contrast, SAP102 was a very poor substrate for all the MAPKs tested (Table I). Table 1. Comparison of substrate specificities of different MAP kinase family members Kinase (0.5 U/ml) Rates of phosphorylation relative to myelin basic protein (MBP) SAP97 SAP102 SAP90 MBP SAPK3/p38γ 100±3 3.1±0.1 100±4 100 SAPK4/p38δ 42±2 0.1 74±5 100 p38α 8.5±0.9 0.1 2.8±0.2 100 p38β 6.3±0.7 0.1 3.1±0.2 100 MAPK2/ERK2 1.7±0.2 0.1 20±5 100 Kinase (0.1 U/ml) Rates of phosphorylation relative to the activating transcription factor-2 (ATF2) SAP97 SAP102 SAP90 ATF2 SAPK3/p38γ 100±2.8 3.1±0.1 100±5 100 JNK2α 1.0±0.2 0.2 6.2±1 100 JNK3 0.5±0.02 1.0±0.1 4.0±0.5 100 JNK1α 0.23±0.1 0.1 0.4±0.1 100 Each enzyme was assayed under initial rate conditions as described previously (Cuenda et al, 1997). The final concentration of the different substrates was 1 μM. SAP97/hDlg was phosphorylated in vitro by SAPK3/p38γ and SAPK4/p38δ at six residues. Three of these (Ser431, Ser442 and Ser447) are located between PDZ domains 2 and 3, whilst residues Ser122, Ser158 and Thr209 are located N-terminal to PDZ domain 1 (Figure 2A). To examine whether endogenous SAP97/hDlg became phosphorylated when cells were exposed to hyperosmotic stress that triggers the activation of p38s, we generated five different phospho-specific antibodies that recognise the major sites phosphorylated by SAPK3/p38γ or SAPK4/p38δ in SAP97/hDlg (Supplementary Figure 1). Endogenous SAP97/hDlg from HEK293 cells became phosphorylated at Ser158, Thr209, Ser431 and Ser442 after osmotic shock, whereas UV-C radiation only induced phosphorylation at Ser158 and Ser442 (Figure 2B). Ser122 was phosphorylated in unstimulated cells and phosphorylation did not increase in response to osmotic stress. The antibody did not recognised SAP97/hDlg in which Ser122 was mutated to Ala (Figure 2C) confirming that this residue is constitutively phosphorylated in cells. Figure 2.Hyperosmotic stress induces phosphorylation of endogenous SAP97/hDlg. (A) Identification of the sites on SAP97/hDlg phosphorylated in vitro by SAPK3/p38γ or SAPK4/p38δ. Rat GST-SAP97 was incubated for 1 h at 30°C with Mg[γ-32P]ATP in the presence of 0.5 U/ml of SAPK3/p38γ or SAPK4/p38δ, and subjected to SDS–PAGE. The phosphorylated SAP97 was excised from the gel, digested with trypsin and the peptides separated by chromatography. The column was developed with an acetonitrile gradient (broken line) and 32P-radioactivity is shown in full line. The phosphopeptides P1–P4 are indicated. To identify the residue phosphorylated in P2, it was subdigested with the protease Asp-N to give a smaller phospho-peptide (residues 427–445). P3 and P4 are a mixture of two peptides, each phosphorylated at a single residue. All residues were identified by a combination of techniques MALDI-TOF, Q-TOF, MS/MS, solid phase sequencing and phospho-amino-acid analysis. (B) Phosphorylation of SAP97/hDlg in HEK293 cells after cellular stress. Cells were incubated for 1 h with or without 10 μM SB203580 and/or 5 μM PD184352, then exposed for 15 min to 0.5 M sorbitol or to UV-C radiation (200 J/m2), followed by a 30 min incubation. Endogenous SAP97/hDlg was immunoprecipitated from 1–5 mg of cell lysate, the pellets immunoblotted using an antibody that recognises SAP97 phosphorylated at S158 (Phos-Ser158), T209 (Phos-Thr209), S431 (Phos-Ser431), S442 (Phos-Ser442) and an antibody that recognises unphosphorylated and phosphorylated SAP97 equally well. The lanes in this panel are duplicates. (C) HEK293 were transfected with SAP97 WT or GST-SAP97 mutant in which S122 has been mutated to Ala. GST-SAP97 was immunoprecipitated from 50 μg of lysate, and the pellets were immunoblotted using Phos-Ser122 antibody. (D) HEK293 cells were incubated for 1 h with or without 400 μM TatSAPK3C(WT) or TatSAPK3C(AA) peptide, and then exposed for 15 min to 0.5 M sorbitol. The immunoprecipitated SAP97/hDlg was immunoblotted as above. Download figure Download PowerPoint In order to obtain information about the kinase(s) responsible for SAP97/hDlg phosphorylation, we incubated cells with SB203580 and/or PD184352, prior to exposure to UV-C or osmotic shock. SB203580 is a relatively specific inhibitor of p38α and p38β activity, whilst PD184352 is a potent inhibitor of the classical ERK1/2 pathway and the ERK5 pathway (Davies et al, 2000; Mody et al, 2001). Neither compound had a significant effect on the phosphorylation of any of the phosphorylation sites of endogenous SAP97/hDlg by these cellular stresses (Figure 2B). In contrast, SB203580 prevented the phosphorylation of Hsp27 a well known in vivo substrate of MAPKAP-K2 which is activated downstream of p38α (Supplementary Figure 2). These results were consistent with phosphorylation of SAP97/hDlg being mediated by SAPK3/p38γ, since UV-C and sorbitol treatment did not activate SAPK4/p38δ in these cells (Supplementary Figure 2), the other MAPK family member that phosphorylates SAP97/hDlg efficiently in vitro and is not inhibited by SB203580. We also incubated cells with the cell permeant peptide TatSAPK3C which contains the last nine residues of SAPK3/p38γ fused to the cell-membrane transduction domain of the human immunodeficiency virus-type 1 (HIV-1) Tat protein. This peptide specifically blocks the phosphorylation of PDZ domain-containing proteins by SAPK3/p38γ in intact cells by preventing the association of the kinase with the PDZ domain of the substrate (Sabio et al, 2004). We treated the cells with wild-type TatSAPK3C(WT) peptide or with the non-interacting mutant TatSAPK3C(AA) peptide, in which the last four residues corresponding to the C-terminal of SAPK3/p38γ are mutated to Ala before exposure to osmotic shock. Phosphorylation of endogenous SAP97/hDlg at all residues induced by exposure to sorbitol was completely suppressed by Tat-SAPK3C(WT), but not by TatSAPK3C(AA) (Figure 2D). The Tat-SAPK3C peptide did not prevent the activation of SAPK3/p38γ or p38α, and did not affect the phosphorylation of MAPKAP-K2 (Supplementary Figure 2). These results indicate that the Tat-SAPK3C peptide prevents SAP97/hDlg phosphorylation by blocking its association with SAPK3/p38γ and again suggest that this kinase is responsible for the phosphorylation of SAP97/hDlg under these conditions. Generation of SAPK3/p38γ and SAPK4/p38δ knockout mice To further investigate whether SAPK3/p38γ mediates SAP97/hDlg phosphorylation in cells we generated mice deficient in SAPK3/p38γ, SAPK4/p38δ or both kinases. To generate these knockout mice we used the targeting constructs shown in Figure 3. ES cells containing the targeted genes were identified by the appearance of 8 kb band on Southern blots when using 3′ probe, in addition to the 11 kb WT band (Figure 3A). The heterozygous SAPK4/p38δ ES cells containing the targeted genes were identified by the appearance of a 7.3 kb band on Southern blots when using 5′ probe, in addition to the 20 kb WT band (Figure 3B). Full details of how the mouse knockouts were generated are previously described (Wiggin et al, 2002). Both, the SAPK3/p38γ and SAPK4/p38δ, knockout mice were viable and fertile and of normal appearance and had no obvious health problems when kept under stress free and specific-pathogen-free conditions. Since SAPK3/p38γ is localised to chromosome 22q (Goedert et al, 1997b) and SAPK4/p38δ to chomosome 6p (Herbison et al, 1999), SAPK3/p38γ and SAPK4/p38δ double knockout were produced by intercrossing mice with single knockouts of each protein kinase. Genotypes of the single and double knockouts were confirmed by PCR (Figure 3A and B). Like the single knockout, the double knockout was viable and fertile and had no obvious health problems. Figure 3.Generation of SAPK3/p38γ and SAPK4/p38δ knockouts. Diagram illustrating the targeting vector for the knockout of SAPK3/p38γ (A) and SAPK4/p38δ (B). The black boxes represent exons, the positions of the probes and the PCR primers used for genotyping are indicated by white boxes and black arrows, respectively. Genomic DNA purified from indicated ES cell lines were digested with BamHI, electrophoresed on a 1% agarose gel, transferred to nitrocellulose for Southern blotting. Genomic DNA purified from tail biopsy sample was used as a template for PCR, electrophoresed on a 1% agarose gel and examined by ethidium bromide staining. (C) Lysates from MEF WT, SAPK3/p38γ, SAPK4/p38δ and SAPK3/4 double knockout (30–50 μg of protein) were immunoblotted with antibodies that recognise specifically each p38. The lanes in this panel are duplicates. (D) MEF SAPK3/p38γ, SAPK4/p38δ and SAPK3/4 double knockout were exposed for 15 min to 0.5 M sorbitol. To examine the activation of p38s, SAPK3/p38γ or SAPK4/p38δ were immunoprecipitated from 2 mg of cell lysates, and immunoblotted with the p38α phospho-specific antibody that also recognises phosphorylated SAPK3/p38γ and SAPK4/p38δ. Alternatively, 50 μg of cell lysates was immunoblotted with the same phospho-specific antibody to detect active p38α. Download figure Download PowerPoint SAPK3/p38γ was expressed at similar levels in the WT and in the SAPK4/p38δ knockout mouse embryonic fibroblasts (MEF) but was not detectable in the SAPK3/p38γ or double knockout MEF (Figure 3C). SAPK4/p38δ was at similar levels in the WT and in the SAPK3/p38γ knockout MEF, but was not detectable in the SAPK4/p38δ or double knockout MEF. The levels of expression of p38α and p38β in either the single or the double knockout were similar to those for WT MEF (Figure 3C). On the other hand, treatment of MEF with cellular stresses caused the activation of the p38 pathway in these cells. In WT, SAPK3/p38γ(−/−), SAPK4/p38δ(−/−) and double knockout cells, p38α was phosphorylated after exposure to osmotic shock (sorbitol), whereas SAPK3/p38γ was activated in WT and SAPK4/p38δ knockout cells, and SAPK4/p38δ was phosphorylated in WT and SAPK3/p38γ knockout cells (Figure 3D). Phosphorylation of SAP97/hDlg in MEF We carried out experiments using MEF from these mice in combination with the use of different kinase inhibitors. In WT MEF, osmotic shock caused detectable phosphorylation of endogenous SAP97/hDlg at Ser158, Thr209 and Ser442, but not Ser431 (Figure 4). When cells were pre-incubated with SB203580 and/or PD184352, neither compound had a significant effect on the phosphorylation of any of the sites of endogenous SAP97/hDlg by sorbitol (Figure 4). Similar results were obtained in SAPK4/p38δ(−/−) fibroblasts, except that the phosphorylation of Thr209 was slightly enhanced compared to the WT (Figure 4B). Figure 4.Phosphorylation of endogenous SAP97/hDlg in mouse embryonic fibroblasts. MEF from WT, SAPK3/p38γ(−/−) (A), SAPK4/p38δ(−/−) (B) or SAPK3/4 double knockout mice (C) were incubated for 1 h with or without 10 μM SB203580 or 5 μM PD184352, then exposed for 15 min to 0.5 M sorbitol. Endogenous SAP97 was immunoprecipitated from 1–5 mg of cell lysate, the pellets immunoblotted using an antibody that recognises SAP97 phosphorylated at S158 (Phos-Ser158), T209 (Phos-Thr209) or S442 (Phos-Ser442), or with an antibody that recognises both unphosphorylated and phosphorylated SAP97. (D) Mouse embryonic fibroblasts from WT, SAPK3/p38γ(−/−) or SAPK4/p38δ(−/−) were incubated for 1 h with or without 400 μM TatSAPK3C(WT) or TatSAPK3C(AA), and then exposed for 15 min to 0.5 M sorbitol. The immunoprecipitated SAP97 was immunoblotted as above. Download figure Download PowerPoint In SAPK3/p38γ(−/−) cells the phosphorylation of SAP97/hDlg at Ser158 was completely lost indicating that this kinase is solely responsible for the phosphorylation of this site (Figure 4A). However, surprisingly endogenous SAP97/hDlg was still phosphorylated at residues Thr209 and Ser442 in SAPK3/p38γ(−/−) cells, although Ser442 phosphorylation was slightly reduced compared to the WT. Moreover, in contrast to WT MEFs, phosphorylation at Ser442 was now blocked if SAPK3/p38γ(−/−) cells were pre-incubated with SB203580 (Figure 4A). However, phosphorylation at Thr209 was unaffected by SB203580 in SAPK3/p38γ(−/−) cells. On the other hand, in double knockout MEF cells deficient in both SAPK3/p38γ and SAPK4/p38δ, the phosphorylation of Thr209 was greatly reduced, and the phosphorylation at Ser442 partially reduced. Phosphorylation at both sites disappeared completely when these cells were pre-treated with SB203580 (Figure 4C). As expected from the results with SAPK3/p38γ(−/−) MEF, the phosphorylation of Ser158 did not occur in the double knockout cells, even in the absence of SB203580. The compound PD184352 did not block the phosphorylation of endogenous SAP97/hDlg by these cellular stresses in either WT, single or double knockout MEF (Figure 4). In order to obtain more information about the protein kinases acting on Thr209 and Ser442, we pre-incubated WT, SAPK3/p38γ(−/−) and SAPK4/p38δ(−/−) MEF with either TatSAPK3C(WT) or with the non-interacting TatSAPK3C(AA) peptide prior to stimulation with sorbitol. In WT and SAPK4/p38δ(−/−) cells, phosphorylation of endogenous SAP97/hDlg at Thr209 and Ser442, was abolished by TatSAPK3C(WT) (Figure 4D). However, in SAPK3/p38γ(−/−) cells, phosphorylation of SAP97/hDlg at both residues, was unaffected by either TatSAPK3C(WT) or TatSAPK3C(AA) peptide (Figure 4D). Taken together our results demonstrate that in WT cells, the phosphorylation of Ser158, Thr209 and Ser442 is mediated by SAPK3/p38γ. In SAPK3/p38γ(−/−) cells SAP97/hDlg is still phosphorylated at Thr209 by SAPK4/p38δ and at Ser442 mainly by p38α or p38β MAPK. Our results are a clear example of functional compensation by highly related protein kinases, when one member is not expressed. Regulation of SAP97/hDlg-GKAP interaction by phosphorylation after hyperosmotic stress Since SAP97/hDlg is a scaffolding protein implicated in the assembly of macromolecular protein complexes we studied whether its phosphorylation affects the binding to other proteins. For this, we first examined the association of SAP97/hDlg with different proteins, such as CASK and GKAP, and the PDZ binding kinase (PBK) and SAPK3/p38γ (Figure 5A), whose association with different domains of SAP97/hDlg is well characterised (Gaudet et al, 2000; Wu et al, 2000; Lee et al, 2002). In co-immunoprecipitation experiments only the interaction of SAP97/hDlg with the protein GKAP is affected by osmotic shock in HEK293 cells (Figure 5A). The anti-SAP97 antibody immunoprecipitated more than 90% of SAP97/hDlg and GKAP from PC12 cells and approx. 50% from HEK293 cells lysates (Figure 5B), suggesting that almost all endogenous GKAP is associated with SAP97/hDlg in these cells. We then checked whether the association of GKAP with SAP97/hDlg in vitro is dependent on the phosphorylation state of SAP97/hDlg. Recombinant SAP97/hDlg either unphosphorylated or phosphorylated by SAPK3/p38γ, was incubated with GKAP. After immunoprecipitation of GKAP the unphosphorylated SAP97/hDlg was detected in the immunocomplex whereas more than 90% of the phospho-SAP97 remained in the supernatant. Moreover, when SAP97 was immunoprecipitated, GKAP was pulled down with the unphosphorylated but not the phosphorylated form of SAP97 (Figure 5C), indicating that the association between these two proteins was regulated by the phosphorylation of SAP97/hDlg. Figure 5.Association of SAP97/hDlg with the protein GKAP is regulated by phosphorylation. (A) Structural organisation of SAP97/hDlg, indicating that the protein CASK binds to the L27 domain, SAPK3/p38γ to the PDZ1 and PDZ3 domains, PBK to PDZ2 and GKAP to the GK domain. HEK293 cells were transfected with PBK or SAPK3/p38γ. Cells were left unstimulated or exposed for 15 min to 0.5 M sorbitol or to UV-C radiation (200 J/m2), followed by a 30 min incubation, and endogenous SAP97/hDlg was immunoprecipitated from 0.2–15 mg of cell lysate. The pellets were immunoblotted using antibodies that recognise CASK, PBK, SAPK3/p38γ, GKAP or SAP97/hDlg. (B) Endogenous SAP97/hDlg was immunoprecipitated from 0.2 mg of undifferentiated PC12 or 15 mg of HEK293 cell lysate. The pellet and 100 μg of protein from both, total lysates (T) (as loading control) or the supernatants (Sup.) were immunoblotted, using antibodies that recognise GKAP or SAP97/hDlg (upper panel). Quantification of" @default.
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