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Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

2003; Springer Nature; Volume: 22; Issue: 6 Linguagem: Inglês

10.1093/emboj/cdg127

1460-2075

Stefan Uhle, Ohad Medalia, Richard T. Waldron, R Dumdey, Peter Henklein, Dawadschargal Bech‐Otschir, Xiaohua Huang, Matthias Berse, Joseph M. Sperling, Rüdiger Schade, Wolfgang Dubiel,

Ubiquitin and proteasome pathways

Article17 March 2003free access Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome Stefan Uhle Stefan Uhle Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Ohad Medalia Ohad Medalia Department of Structural Biology, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Richard Waldron Richard Waldron Department of Medicine, Division of Digestive Diseases, UCLA School of Medicine, Los Angeles, CA, 90095-1786 USA Search for more papers by this author Renate Dumdey Renate Dumdey Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Peter Henklein Peter Henklein Institute of Biochemistry, , Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Dawadschargal Bech-Otschir Dawadschargal Bech-Otschir Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Xiaohua Huang Xiaohua Huang Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Matthias Berse Matthias Berse Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Joseph Sperling Joseph Sperling Department of Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Rüdiger Schade Rüdiger Schade Institute of Pharmacology and Toxicology, Medical Faculty Charité, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Wolfgang Dubiel Corresponding Author Wolfgang Dubiel Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Stefan Uhle Stefan Uhle Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Ohad Medalia Ohad Medalia Department of Structural Biology, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Richard Waldron Richard Waldron Department of Medicine, Division of Digestive Diseases, UCLA School of Medicine, Los Angeles, CA, 90095-1786 USA Search for more papers by this author Renate Dumdey Renate Dumdey Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Peter Henklein Peter Henklein Institute of Biochemistry, , Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Dawadschargal Bech-Otschir Dawadschargal Bech-Otschir Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Xiaohua Huang Xiaohua Huang Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Matthias Berse Matthias Berse Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Joseph Sperling Joseph Sperling Department of Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Rüdiger Schade Rüdiger Schade Institute of Pharmacology and Toxicology, Medical Faculty Charité, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Wolfgang Dubiel Corresponding Author Wolfgang Dubiel Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany Search for more papers by this author Author Information Stefan Uhle1, Ohad Medalia2, Richard Waldron3, Renate Dumdey1, Peter Henklein4, Dawadschargal Bech-Otschir1, Xiaohua Huang1, Matthias Berse1, Joseph Sperling5, Rüdiger Schade6 and Wolfgang Dubiel 1 1Division of Molecular Biology, Department of Surgery, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany 2Department of Structural Biology, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany 3Department of Medicine, Division of Digestive Diseases, UCLA School of Medicine, Los Angeles, CA, 90095-1786 USA 4Institute of Biochemistry, , Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany 5Department of Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel 6Institute of Pharmacology and Toxicology, Medical Faculty Charité, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1302-1312https://doi.org/10.1093/emboj/cdg127 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipi tation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation. Introduction The COP9 signalosome (CSN) is a multimeric complex that is conserved from yeast to man (Deng et al., 2000). Since its discovery in plant cells (Wei et al., 1994), a function in signaling and developmental processes has been implicated. In fact, the complex is a negative regulator of photomorphogenesis (for a review, see Wei and Deng, 1999) and is involved in development of Drosophila (Freilich et al., 1999). However, the exact function of the CSN has not been elucidated yet. Purification and characterization of the CSN from mammalian cells revealed sequence homologies between CSN subunits and components of the 26S proteasome lid complex (Seeger et al., 1998; Wei et al., 1998), as well as components of the eukaryotic translation initiation factor 3 (eIF3) complex (Glickman et al., 1998). Structural similarities and co-purification (Kapelari et al., 2000) of the CSN and the lid suggested a functional relationship between the CSN and the ubiquitin (Ub)/26S proteasome system. Indeed, the CSN is involved in regulation of the stability of proteins such as p27 (Tomoda et al., 1999), c-Jun (Naumann et al., 1999), p53 (Bech-Otschir et al., 2001) and HY5 (Hardtke et al., 2000), which are substrates of the Ub/26S proteasome system. Recently, an effect of the CSN on the activity of Ub ligases termed SCF (SKP1–CDC53–F-box protein) complexes has been shown. The SCF Ub ligases collaborate with specific Ub conjugating enzymes in the ubiquityl ation of different substrates. One component of the SCF complexes is a member of the cullin (Cul) protein family, which is covalently modified by the Ub-like protein NEDD8. The conjugation of NEDD8 to Cul1 enhances the recruitment of Ub conjugating enzyme Ubc4 to the SCF complex, which stimulates protein polyubiquitylation (Kawakami et al., 2001). It has been demonstrated in vitro and in vivo that the CSN removes NEDD8 from Cul1 (Lyapina et al., 2001; Schwechheimer et al., 2001; Zhou et al., 2001). The responsible deneddylation activity seems to be localized in the MPN domain of CSN5 (Cope et al., 2002). Although data on the effect of CSN-mediated deneddylation on SCF-dependent substrates are controversial, reduction of the SCF Ub ligase activity by NEDD8 removal is very likely, as it has been shown for pcu3/Cul-3 complexes in Schizosaccharomyces pombe (Zhou et al., 2001) and for the SCF complex involved in p27 ubiquitylation (Yang et al., 2002). The CSN from human red blood cells co-purifies with kinase activity, which phosphorylates IκBα, c-Jun, p53 and interferon consensus sequence binding protein (ICSBP) (for a review, see Bech-Otschir et al., 2002). Because none of the CSN subunits possesses sequence homologies with protein kinases, an associated kinase activity has been assumed. Phosphorylation of p53 by the CSN-associated kinase activity targets the tumor suppressor to degradation by the Ub pathway. Inhibition of p53 phosphorylation by curcumin, an inhibitor of the CSN-associated kinases (Henke et al., 1999), leads to stabilization of the endogenous tumor suppressor in tumor cells (Bech-Otschir et al., 2001). The transcription factor c-Jun is stabilized towards the Ub/26S proteasome system upon phosphorylation by CSN-associated kinase (Naumann et al., 1999). Overexpression of the CSN subunit 2 (CSN2) causes a significant increase of activation factor 1 (AP1) transactivation activity. Under these conditions c-Jun, a major component of AP1, is stabilized in HeLa cells, which is accompanied by de novo assembly of the CSN complex. The CSN-directed c-Jun signaling controls a major portion of vascular endothelial growth factor production in tumor cells (Pollmann et al., 2001). The transcription factor HY5 is a positive regulator of light-regulated genes in plant cells. It is degraded in the dark by the Ub system. For this process the CSN as well as the autonomous repressor of photomorphogenesis COP1 is required (for a review, see Schwechheimer and Deng, 2001). COP1 has been suggested to be the responsible Ub ligase of HY5 (Osterlund et al., 2000). Binding of HY5 to COP1 is prevented by light-regulated phosphorylation of HY5 presumably by the protein kinase CK2 (CK2, formerly casein kinase II), which stabilizes the transcription factor in the light (Hardtke et al., 2000). Recently, the co-purification of the inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) with the CSN (Wilson et al., 2001; Sun et al., 2002) has been reported. The enzyme is sensitive to curcumin and can act as a protein kinase that phosphorylates c-Jun and ATF-2. Here we show the co-purification and association of the protein kinase D (PKD) and CK2 with the CSN. We demonstrate that the two kinases phosphorylate c-Jun as well as p53. In addition, the two kinases are responsible for CSN subunit modification. We provide data indicating an impact of the kinases on ubiquitylation and degradation of c-Jun. Results CK2 and PKD co-purify with the CSN To identify CSN-associated kinases we studied phosphopeptide analyses performed with c-Jun (Seeger et al., 1998), p53 (Bech-Otschir et al., 2001), ICSBP (Cohen et al., 2000) and IκBα. Experiments were perfomed with recombinant substrates of CSN-associated kinases, with purified CSN from human erythrocytes and with [γ-32P]ATP. Phosphorylated proteins were gel purified, digested with chymotrypsin and peptides were separated by high-performance liquid chromatography (HPLC). Radioactive, phosphorylated peptides identified by peptide sequencing and mass spectrometry are shown in Table I in order of their specific radioactivity. Most of the peptides contain serines and threonines as putative phosphorylation sites, suggesting that CSN is associated with Ser/Thr kinases. Table 1. Peptides phosphorylated by the CSN-associated kinases identified by phosphopeptide analyses with different proteins Protein Radioactive peptides Putative P sites Consensus c-Jun 58SDLLTSPDVGLLKLASPELERL79 Ser63, Ser73 JNK Thr62 CK2 115VRALAELHSQNTLPSVTSAAQ135 ? No 53LRAKNSDLLTSPDVGLL69 Ser63 JNK Thr62 CK2 151GGSGSGGFSASLHSEPPVYANLSNF177 ? No 80IIQSSNGHITTTPTPTQF97 ? No 98LCPKNVTDEQEGFAEGF114 ? No 36QSMTLNLADPVGSLKPH52 Ser48 PKC p53 142PVQLWVDSTPPPGTRVRA159 Thr155 No 94SSSVPSQKTY103 Ser99 PKC 384MFKTEGPDSD393 Ser392 CK2 ICSBP 253PPADAIPSERQRQVTRK269 Ser260 PKC 93EEVTDRSQLDISEPYK108 Ser99 CK2 IκBα 271QLTLENLQMLPESEDEESYDTESEFT296 Ser283 CK2 Thr291 CK2 ICSBP, interferon consensus sequence binding protein; JNK, Jun-N-terminal kinase. Consensus sequences for CK2 or PKC phosphorylation are indicated in bold. Unknown phosphorylation sites are indicated by ‘?’. The c-Jun peptide with the highest specific radioactivity contains Ser63 and Ser73, known to be phosphorylation sites of the Jun-N-terminal kinase (JNK; Musti et al., 1997). However, immunoblots with purified CSN using different antibodies against different JNK isoforms revealed that these kinases were not detectable in our preparations (data not shown). Interestingly, the same c-Jun peptide 58–79 contains a consensus sequence for CK2 phosphorylation (see below). In addition, other radiolabeled peptides also exhibit putative CK2 phosphorylation sites. Immunoblots and immunoprecipitations revealed that CK2 is a CSN-associated kinase (see below). A number of peptides contained protein kinase C (PKC) sites (see Table I). Antibodies against PKCα, β, γ and θ indicated that these PKC isoforms were not detectable in CSN preparations (data not shown). On the other hand, an antibody against PKCμ, also known as PKD, identified PKD in our CSN preparations. To investigate whether PKD and CK2 co-purify with the CSN, immunoblots on different CSN preparation steps were performed. The data are summarized in Figure 1. Figure 1A shows western blots with fractions of a DEAE column, a very early step of the preparation. Many fractions containing the CSN, as indicated by the anti-CSN5 antibody, also contained PKD and CK2α, a subunit of CK2. In addition, the 5/6-kinase co-eluted with CSN fractions. Fractions 30–40 were pooled for further CSN purification. The next step was a 10 to 40% glycerol gradient in which the CSN, PKD, CK2 and 5/6-kinase co-sedimented to similar fractions (Figure 1B). Western blots with fractions from a ResourceQ column are shown in Figure 1C. CK2 was detected in CSN fractions eluted with high salt. PKD was found in most of the CSN5-containing fractions. For further purification we used fractions 36–43. Fractions eluted with lower salt contained 5/6-kinase (Figure 1C). After the ResourceQ column the 5/6-kinase was not detectable in our CSN pool. The last step was a MonoQ ion exchange chromatography. Purified CSN, which was obtained in MonoQ fractions 11 and 12, is shown in Figure 1D. As demonstrated by immunoblotting, the same fractions contained PKD and CK2α, but not 5/6-kinase. Figure 1.CK2 and PKD co-purify with the CSN during its preparation from human erythrocytes. (A) Western blots with fractions 21–47 from DEAE column (Seeger et al., 1998). The blots were probed with anti-PKD, anti-5/6-kinase, anti-CK2α and anti-CSN5 antibodies. Fractions 30–40 indicated by arrows were pooled for determination of specific curcumin-sensitive kinase activity [see (E) and Materials and methods] and for further purification. (B) Western blots with fractions 3–18 from a 10 to 40% glycerol gradient using same antibodies as in (A). Fractions 11–14 (arrows) were pooled for further use. (C) Western blots with fractions 28–45 obtained from a RecourceQ column using the same antibodies as in (A). Fractions 36–43 were pooled. (D) SDS–PAGE of 20-μl aliquots of fractions 11 and 12 after MonoQ ion exchange chromatography. Core CSN subunits are indicated. Western blots with the same fractions using anti-PKD, anti-5/6-kinase and anti-CK2α antibodies revealed the presence of CK2α and PKD, but not of 5/6-kinase, in the final CSN preparation. (E) Curcumin-sensitive kinase activity co-purifies with the CSN. Fixing the kinase activity in the lysate to 1, a 18 000-fold co-purification of CSN-associated kinase activity was achieved by the final preparation steps. Download figure Download PowerPoint During CSN preparation the specific curcumin-sensitive phosphorylation of c-Jun was measured in pooled fractions indicated by arrows (see Figure 1A–C). Curcumin is an inhibitor of CSN-associated kinases and the specific curcumin-sensitive kinase activity should, at least in part, reflect co-purification of associated kinase activity. The data are summarized in the table of Figure 1E. Compared with cell lysate, the curcumin-sensitive kinase activity per microgram of protein was 1500-fold higher in the pooled DEAE fractions. Further significant enrichment of specific curcumin-sensitive kinase in pooled CSN fractions was obtained by glycerol gradient centrifugation. Although specific curcumin-sensitive kinase activity increased from the glycerol gradient to ResourceQ column, 5/6-kinase disappeared from our CSN pool after ResourceQ. There was no further increase of specific curcumin-sensitive kinase activity by MonoQ. CK2 and PKD are associated with the CSN Because CSN subunits do not possess consensus sequences for ATP binding, a possible interaction of ATP with the CSN should be mediated by associated proteins, most likely by the associated kinases. Initial cross-linking experiments with perjodate-oxidized ATP indicated that ATP binds to the CSN (data not shown). Therefore, a gold-labeled non-cleavable ATP analog was incubated with the CSN and the mixture was analyzed by electron microscopy. The data are shown in Figure 2A. Approximately 10–15% of all CSN particles were estimated to be labeled with gold-ATP. As seen in a gallery of gold-ATP-labeled CSN particles (right panel), there was one gold particle associated with the periphery of one CSN complex. Because our CSN preparation did not contain detectable 5/6-kinase, ATP-binding cannot be explained by this enzyme. Figure 2.CK2 and PKD are associated with the CSN. (A) Electron microscopy of CSN particles that were incubated with gold-labeled uncleav able ATP analog. The gallery (right panel) shows typical positions of gold clusters mediated by kinases associated with CSN complexes. (B) Immunoprecipitation of the purified CSN was performed with anti-CSN7 antibody. In the left panel purified CSN and recombinant Myc-tagged PKD were mixed and the mixture was immunoprecipitated (IP: anti-CSN7). The western blot revealed the presence of CSN5 (anti-CSN5) and of Myc-tagged PKD in the precipitate. Myc-PKD is the recombinant PKD alone and for control immunoprecipitation protocol was performed without anti-CSN7 antibody, indicating unspecific binding to protein A–Sepharose. In the right panel, purified CSN shown in Figure 1D was immunoprecipitated (IP: anti-CSN7). The two lanes of western blots show two different immunoprecipitations and indicate the presence of CSN5 and of CK2α in the precipitate. The control was performed without anti-CSN7 antibody. (C) Immunoprecipitation of the CSN from HeLa cell lysate using the anti-CSN7 antibody. The left panel shows immunoblots of the precipitates (IP: anti-CSN7). The blot was probed with anti-PKD, anti-Cul1, anti-CK2α and anti-CSN5 antibodies. Control experiments were performed as in (B). Immuno precipitation with pre-immune serum (IP: pre-immune) indicates the specificity of immunoprecipitations. The right panel shows autoradiography of an SDS–PAGE carried out with the reaction mixtures of kinase assays. The anti-CSN7 immunoprecipitate was used as a source of kinase activity and was incubated in the presence of IκBα or c-Jun as a substrate, [γ-32P]ATP, and with or without curcumin. To demonstrate the specificity of the kinase reaction, the ability of anti-CSN7 immunoprecipitate to phosphorylate recombinant CSN7 or CSN6 was tested. Depending on the exposure time, phosphorylation of heavy chain rabbit IgG was seen (IgG). Download figure Download PowerPoint To test whether CK2 and PKD are associated with the CSN, immunoprecipitations were carried out using an anti-CSN7 antibody. Purified CSN and recombinant Myc-tagged PKD were incubated at 37°C and after 30 min immunoprecipitation was performed (Figure 2B). The western blot with the anti-CSN5 antibody indicated that the CSN complex was in the precipitate. In addition, PKD co-immunoprecipitated under these conditions. In the case of CK2, purified CSN shown in Figure 1D (MonoQ fraction 12) was immunoprecipitated with anti-CSN7 antibody. Western blot analysis of the precipitate revealed that CK2α is associated with the CSN (Figure 2B, right panel). Immunoprecipitations were also performed with HeLa cell lysate (Figure 2C). HeLa cells were used because most of our cell experiments were performed with this cell line. As indicated by the anti-CSN5 antibody, endogenous HeLa cell CSN was successfully precipitated. The precipitate also contained Cul1, which has been shown to interact with the CSN (Lyapina et al., 2001; Schwechheimer et al., 2001). In addition, both PKD and CK2α were co-immunoprecipitated. We were interested to determine whether the CSN from HeLa cells is associated with active kinases. Therefore, the anti-CSN7 immunoprecipitate was used in kinase assays with IκBα or c-Jun as substrate. As shown in Figure 2C (right panel), IκBα was phosphorylated under this condition. As estimated by densitometry, up to 80% of the IκBα phosphorylation was inhibited by curcumin. In addition, the anti-CSN7 immunoprecipitate phosphorylated c-Jun in a curcumin-sensitive manner. CSN subunits such as CSN7 are substrates of CSN-associated kinases, whereas others, such as CSN6, are not (see below). To evaluate the specificity of phosphorylation, CSN7 and CSN6 were used as substrates. As shown in Figure 2C (right panel), the anti-CSN7 immunoprecipitate phosphorylated CSN7, but not CSN6. To identify CSN subunits, which interact with CK2 and/or PKD far-western blots with recombinant CSN subunits, the purified CSN complex and with recombinant kinases were carried out. Recombinant kinases were incubated with immobilized recombinant CSN subunits and CSN. After washing, blots were probed with anti-Myc, anti-CK2α or anti-CK2β antibodies. The data are summarized in Figure 3. Recombinant Myc-tagged PKD showed an interaction with full-length CSN3 subunit of the purified CSN. Under these conditions only very weak inter action with recombinant ΔCSN3(111–403) was detected (Figure 3B). Figure 3.Far-western blots indicate that CK2 and PKD bind to subunits of the CSN. (A) The Ponceau stain of nitrocellulose shows selected recombinant CSN subunits used in far-western blots. A Coomassie Blue-stained SDS–PAGE demonstrates the purified CSN, which was immobilized on nitrocellulose. (B) Recombinant N-terminal Myc-tagged PKD binds to full-length CSN3 of the purified CSN and very weak to ΔCSN3(111–403), as indicated by the anti-Myc antibody. Immobilized recombinant CSN7 is shown as negative control. (C) Immobilized recombinant CSN subunits were incubated with recombinant CK2 consisting of 2α2β. After washing the blots were probed with an anti-CK2α (left panel) or an anti-CK2β antibody (right panel). CSN6 is shown as a negative control. Download figure Download PowerPoint In Figure 3C it is demonstrated that CK2 interacts with ΔCSN3(111–403) and CSN7. The anti-CK2α antibody shows a strong reaction with ΔCSN3(111–403) fragment, but weakly interacts with recombinant CSN7. In contrast, anti-CK2β antibody indicates strong interaction with CSN7. Its reaction with ΔCSN3(111–403), however, is very weak. CK2 and PKD phosphorylate CSN subunits, p53 and c-Jun To determine whether the associated kinases are responsible for protein modifications described previously (Bech-Otschir et al., 2002), in vitro kinase assays were performed with recombinant kinases and [γ-32P]ATP. It has been observed previously that CSN subunits are phosphorylated by the CSN-associated kinases (Kapelari et al., 2000) or by other kinases (Karniol et al., 1999). Therefore, recombinant CSN subunits and the purified CSN complex were used as substrates in kinase assays with recombinant CK2 and PKD. The data are summarized in Figure 4. CK2 modified CSN2 and CSN7 as recombinant proteins as well as in the purified complex (Figure 4B). PKD shows weak phosphorylation of recombinant CSN2, CSN5 and CSN7, but has a strong effect on CSN7 in the purified complex (Figure 4C). There are different effects of different proteins on the autophos phorylation of the recombinant kinases. To demonstrate that equal amounts of PKD were added to all samples, a Coomassie Blue-stained gel is shown in Figure 4C (lower panel). To determine whether phosphorylation of CSN subunits occurs in cells, lysate obtained from reticulocytes was incubated with [γ-32P]ATP. After incubation the CSN was immunoprecipitated. As shown in Figure 4D, autoradiography of immunoprecipitated CSN identified a radioactive band, which is most likely identical to CSN2. Under this condition significant phosphorylation of CSN7 was not observed. On the other hand, immunoprecipitated CSN from HeLa cells was able to phosphorylate CSN7 (Figure 2C, right panel). Figure 4.CK2 and PKD phosphorylate subunits of the CSN. (A) In vitro kinase assays were performed with shown recombinant CSN subunits and purified CSN as substrates (Coomassie). (B) Each recombinant CSN subunit or purified CSN were incubated with recombinant CK2. Autoradiography shows phosphorylated recombinant CSN subunits labeled with stars. Autoradiography of the phosphorylated CSN complex reveals that phosphorylated CSN7 and autophosphorylated CK2β co-migrate in SDS–PAGE. Complex-bound subunits migrate faster in SDS–PAGE as compared with recombinant His6-tagged CSN subunits. (C) Kinase reactions were carried out as in (B) using recombinant PKD. Phosphorylated recombinant subunits are indicated by stars. Different proteins exert different effects on autophosphorylation of recombinant kinases. To demonstrate that equal amounts of PKD were added to each sample, Coomassie Blue-stained PKD is shown (lower panel). (D) Subunit CSN2 of endogenous CSN is phosphorylated in reticulocyte lysate. Reticulocyte lysate was incubated with [γ-32P]ATP. After incubation endogenous CSN was immunoprecipitated and the precipitate was analyzed by SDS–PAGE and autoradiography. Control (CSN): purified CSN was incubated with [γ-32P]ATP and then analyzed by SDS–PAGE and autoradiography. Download figure Download PowerPoint Next, p53 and c-Jun were used as substrates of recombinant PKD or CK2. Figure 5A shows that both CK2 and PKD phosphorylated c-Jun as well as p53. In addition, autophosphorylations of CK2α, CK2β and PKD were observed. Figure 5.CK2 and PKD phosphorylate c-Jun and p53. (A) Recombinant c-Jun and p53 shown in the Coomassie Blue-stained gel were used for in vitro kinase assays. CK2: recombinant c-Jun or p53 was incubated with recombinant CK2 in presence of [γ-32P]ATP. After 1 h at 37°C the reaction mix was separated by SDS–PAGE and the dried gel was autoradiographed. The autoradiography shows phosphorylation of c-Jun and p53 as well as autophosphorylation of CK2α and CK2β. PKD: the kinase reaction was carried out with recombinant PKD as described above. The autoradiography shows phosphorylation of c-Jun and p53 as well as autophosphorylation of PKD. (B) Inhibition of CK2-dependent p53 phosphorylation by phosphorylated Δp53(145–164) peptide. Recombinant p53 was incubated with recombinant CK2 as outlined in (A). Phosphorylated Δp53(145–164) was added to the reaction mix at indicated concentrations. (C) CK2 phosphorylates Thr155 of p53. Three different modifications of Δp53(145–164) shown in the upper panel were tested in kinase assays with recombinant CK2. Stars indicate phosphorylated serine (S*) or threonine (T*). After kinase reaction samples were analyzed by 15% SDS–PAGE and autoradiography. (D) Inhibition of CK2-dependent c-Jun phosphorylation by Δc-Jun(59–78). Recombinant c-Jun was incubated with recombinant CK2 as described in (A) and Δc-Jun(59–78) was added at indicated concentrations. Lower panel: Δc-Jun(59–78) was phosphorylated by CK2. Download figure Download PowerPoint We then wished to determine which of the two kinases is responsible for p53 phosphorylation that targets the tumor suppressor to degradation by the Ub system. In previous experiments it has been shown that p53 is phosphorylated at Thr155 by the CSN-associated kinases and that this modification is crucial for p53 stability (Bech-Otschir et al., 2001). In addition, we have shown that the p53 peptide Δp53(145–164) acts as a specific competitor by inhibiting p53 phosphorylation, which stabilizes the tumor suppressor towards degradation by the Ub system in vitro and in vivo (Bech-Otschir et al., 2001). Therefore, the effect of Δp53(145–164) with phosphorylated Ser149, Thr150 and Thr155 on the phosphorylation of p53 by recombinant PKD and CK2 was tested. Phosphorylated Δp53(145–164) did not influence the modification of p53 by PKD (data not shown). In contrast, there was a significant inhibition of p53 phosphorylation by CK2, as shown in Figure 5B. Moreover, Δp53(145–164) was directly phosphorylated by CK2 (Figure 5C). To test whether Thr155 is a target residue of CK