Báo cáo khoa học: Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis doc

Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis Yoshihiko Miyata and Eisuke Nishida Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Japan Keywords Cdc37; CK2; gel electrophoresis; Hsp90; protein kinase Correspondence Y Miyata, Department of Cell & Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Fax: +81 75 753 4235 Tel: +81 75 753 4231 E-mail: ymiyata@lif.kyoto-u.ac.jp (Received 22 March 2007, revised August 2007, accepted September 2007) doi:10.1111/j.1742-4658.2007.06090.x The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem 278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem 278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol 24, 4065–4074] Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg2+ and ATP The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52 Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis Protein kinases play pivotal roles in cellular signal transduction systems Reversible protein phosphorylation is one of the major mechanisms used to control the function, localization and stability of proteins inside cells [1] Therefore, the analysis of protein kinase activity and the phosphorylation level of their substrates are important for understanding signal transduction pathways at a molecular level Many methods have been described for determining protein kinase activity and protein phosphorylation levels In vitro, phosphorylation reactions can be monitored by incubating a protein kinase and a substrate in the presence of radioactive ATP ([32P]ATP[cP]), followed by SDS ⁄ PAGE and autoradiography to quantify radioactivity in the substrate When a peptide substrate is used, the amount of radioactivity incorporated into Abbreviations Cdc37, cell division cycle protein 37; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FKBP52, FK506-binding protein 52; GST, glutathione S-transferase; HRP, horseradish peroxidase; Hsp90, heat shock protein 90; TBB, 4,5,6,7tetrabromobenzotriazole 5690 FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida the peptide can be quantified by scintillation counting after separating the radioactive peptide from free ATP ⁄ ADP using phosphocellulose filters Protein phosphorylation in vivo can be examined in several ways The phosphorylation level of a substrate can be determined by isolating the substrate from radiolabeled cells or tissues by immunoprecipitation followed by SDS ⁄ PAGE and autoradiography For a phosphorylation site with a known sequence, it is possible to obtain a phospho-specific antibody that reacts with the substrate only in its phosphorylated form by immunization and affinity purification with the corresponding phosphopeptide A phospho-specific antibody can then be used to directly quantify the site-specific phosphorylation of a substrate in vivo by western blot analysis Recently, MS has become a powerful technology for large-scale detection and quantification of in vivo protein phosphorylation [2,3] Although the precise molecular mechanism remains to be elucidated, in some cases protein phosphorylation causes a mobility shift of the protein band on SDS ⁄ PAGE, often but not always decreasing the mobility However, the mobility shifts are generally not very large, and in most cases protein phosphorylation does not induce a mobility shift at all Moreover, achieving optimal band shifts often requires special gel compositions (such as a low concentration of bis-acrylamide), which can only be determined by somewhat hit-and-miss experimentation A more reproducible and reliable method for discriminating phosphorylated and nonphosphorylated forms of a broad range of proteins by gel electrophoresis has long been sought Recently, Kinoshita et al identified alkoxide-bridged dinuclear metal complexes as novel phosphate-binding compounds that preferentially capture phosphomonoester dianions bound to serine, threonine and tyrosine residues in proteins [4] They also reported that these compounds could be used to separate phosphorylated and unphosphorylated proteins in SDS ⁄ PAGE [5] Protein kinase activity in cells is regulated in many different ways Releasing an inhibitory subunit from a catalytic subunit can activate a kinase By contrast, binding an activating regulatory coprotein to an inactive catalytic subunit can activate a kinase In many signal-transducing protein kinases, site-specific phosphorylation by an upstream protein kinase (a kinasekinase) activates them Before these activation steps, the protein kinases must be in the correct structural conformation, to be activated by the appropriate stimuli However, the activation-ready structures of signaling protein kinases are relatively unstable in nature and require additional proteins called ‘molecular chaperones’ to stabilize them within cells Among the Cdc37 phosphorylation by CK2 and signaling kinases molecular chaperones, heat shock protein 90 (Hsp90) and cell division cycle protein 37 (Cdc37) have been shown to be specifically required for the stability and function of many signaling protein kinases, including Raf1 [6,7], Cdk4 [8–10], MOK [11], IKK [12], and v-Src [13] Hsp90 is an important molecular chaperone whose ATP-dependent function is essential for the folding and function of many signaling molecules, including protein kinases and steroid hormone receptors [14–16] Cdc37 both acts as a molecular chaperone by itself and is also required for the efficient recruitment of Hsp90 to protein kinase complexes [17,18] Therefore, Cdc37 activity is crucial for many signaling protein kinases to function correctly in vivo Protein kinase CK2 is a ubiquitous and highly conserved protein kinase that is known to be involved in many physiological functions by phosphorylating a plethora of substrates [19–21] CK2 is elevated in many types of tumor, and its overexpression is tumorigenic in experimental models in animals, suggesting that CK2 is involved in both cell cycle control and neoplastic cell growth [22] Although CK2 was one of the earliest protein kinases identified, its regulatory mechanism remains largely unknown CK2 is composed of two catalytic subunits (a and ⁄ or a¢) and two noncatalytic subunits (b) The catalytic subunits of CK2 are constitutively active, whether or not they are associated with noncatalytic b-subunits [23], CK2 activity is independent of any known second messengers, and no upstream ‘kinase-kinase’ has been identified that activates CK2 [24] We and others have previously identified Cdc37 as a pivotal substrate for CK2 and reported that Cdc37 phosphorylation by CK2 is essential for Cdc37 to act as a molecular chaperone for many signaling protein kinases [25–28] Moreover, the molecular chaperone functions of Hsp90 and Cdc37 are required for CK2 itself to be optimally active [25,29,30], suggesting that CK2 and Cdc37 together constitute a positive feedback mechanism to control various signaling protein kinases [25,31] Therefore, analyzing the level of CK2-dependent Cdc37 phosphorylation in vivo should be crucial for understanding the regulatory mechanisms of Cdc37, CK2, and other signaling protein kinases In this report, we describe a new antibody that recognizes Cdc37 only when phosphorylated by CK2 We also demonstrate that the phosphorylation of Cdc37 can be analyzed by phospho-affinity gel electrophoresis Together, the phospho-specific antibody and phospho-affinity gel electrophoresis enabled us to directly study CK2-dependent Cdc37 phosphorylation in vitro and in vivo FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5691 Cdc37 phosphorylation by CK2 and signaling kinases Y Miyata and E Nishida Results Phospho-specific antibody against Cdc37 We synthesized the phosphopeptide VWDHIEVpSDDEDETHC, including amino acid residues 6–20 of mammalian Cdc37, in which Ser13 was phosphorylated This sequence is in the most N-terminal region of Cdc37, where the amino acid sequence conservation between species is highest In fact, the amino acid sequence of the peptide we used is identical in human, chimpanzee, rhesus monkey, cattle, pig, rat, mouse and chick sequences The serine at position 13 of Cdc37, which was included in the synthetic peptide, has been reported to be phosphorylated by CK2 and important for the functional regulation of Cdc37 [25– 28] We raised rabbit antiserum against this phosphopeptide and examined the specificity of the purified antibody Purified recombinant Cdc37 was incubated with the recombinant catalytic subunit of CK2 (CK2a) or with CK2 holoenzyme (CK2a2b2) or alone, in the presence or absence of Mg2+ and ⁄ or ATP As shown in Fig 1A, the antibody (anti-[pSer13]-Cdc37 hereafter) recognized Cdc37 only when incubated with either CK2a or CK2 holoenzyme in the presence of ATP and Mg2+ (lanes and 9) The absence of either CK2 (lane 3), Mg2+ (lanes and 7) or ATP (lanes and 8) completely abolished antibody binding (Fig 1A), indicating that anti-[pSer13]-Cdc37 specifically recognized the CK2-phosphorylated form of Cdc37 The presence of equal amounts of Cdc37 in the phosphorylation mixtures was checked by probing western blots with an antibody to Cdc37 (Fig 1B) The binding of anti-[pSer13]-Cdc37 was completely abolished when Ser13 in Cdc37 was replaced by a nonphosphorylatable amino acid Recombinant wild-type Cdc37 and two Cdc37 mutants in which Ser13 was replaced by alanine [Cdc37(13SA)] or aspartic acid [Cdc37(13SD)] were incubated with CK2a or CK2 holoenzyme or alone, in the presence of Mg2+–ATP Wildtype Cdc37 bound anti-[pSer13]-Cdc37 in the presence of CK2a or CK2 holoenzyme (Fig 1C, lanes and 7) By contrast, neither Cdc37(13SA) nor Cdc37(13SD) were recognized by anti-[pSer13]-Cdc37, even after incubation with CK2a or CK2 holoenzyme (Fig 1C, lanes 5, 6, and 9) The amounts of Cdc37(WT), Cdc37(13SA) and Cdc37(13SD) in all the incubation mixtures were approximately the same, as shown by Coomassie brilliant blue (CBB) staining (Fig 1D) These results showed that anti-[pSer13]-Cdc37 recognized Cdc37 only when Ser13 was phosphorylated by CK2, and that isolated CK2a phosphorylated the same site in Cdc37 (Ser13) as purified CK2 holoenzyme We next examined the specificity of anti-[pSer13]Cdc37 for Cdc37 in comparison to other CK2-phosphorylated proteins CK2 phosphorylates serine or threonine residues followed by a stretch of acidic amino acids [19,21], which constitutes a consensus Fig Phospho-specific antibody against an Hsp90 cochaperone, Cdc37 (A, B) Recombinant Cdc37 was incubated at 30 °C for 30 alone (lanes 1–3), with CK2a (lanes 4–6), or with purified CK2 holoenzyme (lanes 7–9), in the presence (+) or absence (–) of Mg2+ and ⁄ or ATP as indicated above the track Western blots of these mixtures with anti-[pSer13]-Cdc37 (A) or with anti-Cdc37 (B) are shown The positions of molecular weight markers and Cdc37 are shown (C, D) Wild-type protein [Cdc37(WT), lanes 1, and 7] as well as two mutant proteins [Cdc37(13SA), lanes 2, and 8, and Cdc37(13SD), lanes 3, and 9] were incubated at 30 °C for 30 alone (lanes 1–3), with CK2a (lanes 4–6) or with CK2 holoenzyme (lanes 7–9), and the mixtures were analyzed by western blotting with anti-[pSer13]-Cdc37 (C) CBB staining is shown in (D) The positions of molecular weight markers and Cdc37 are shown 5692 FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida phosphorylation sequence for CK2 Thus, the amino acid sequences surrounding CK2 phosphorylation sites are similar in most CK2 substrates We examined two other molecular chaperones, Hsp90 and FK506-binding protein 52 (FKBP52), which are also known to be phosphorylated by CK2, to find whether they were recognized by anti-[pSer13]-Cdc37 when phosphorylated In fact, the amino acid sequences around the known CK2 phosphorylation sites in Cdc37 (Ser13) [26,28], FKBP52 (Thr143) [32], and Hsp90 (Ser231 and Ser263) [33] are highly homologous (Fig 2A) Purified recombinant FKBP52 and Hsp90 were incubated with or without CK2 in the presence of Mg2+– [32P]ATP[cP] Cdc37(WT) and Cdc37(13SA) were included as positive and negative controls Analysis of the phosphorylation mixtures by SDS ⁄ PAGE and autoradiography clearly showed that Cdc37(WT), FKBP52 and Hsp90 were heavily phosphorylated by CK2 in vitro (Fig 2B) Western blot analysis of the same phosphorylation mixtures probed with anti[pSer13]-Cdc37 showed that the antibody only recognized CK2-phosphorylated Cdc37 (Fig 2C, lane 5), and not CK2-phosphorylated FKBP52 (Fig 2C, lane 7) or CK2-phosphorylated Hsp90 (Fig 2C, lane 8) The presence of equal amounts of Cdc37, FKBP52 and Hsp90 in the phosphorylation mixtures were checked by CBB staining (Fig 2D) These results indicated that anti-[pSer13]-Cdc37 specifically recognized the CK2-phosphorylated form of Cdc37, and did not crossreact with a generic CK2 phosphorylation consensus sequence Phosphorylated Cdc37 associates with signaling protein kinases Cdc37 phosphorylation by CK2 and signaling kinases to Cdc37, we next investigated whether the CK2-phosphorylated form of Cdc37 associated with signaling protein kinases in vivo Four typical Hsp90 client kinases, Cdk4, MOK, v-Src, and Raf1, were expressed in COS7 cells as FLAG-tagged proteins, and kinase– Hsp90–Cdc37 complexes were immunopurified on anti-FLAG affinity resin As controls, two nonclient kinases for Hsp90, CK1 and DYRK2, were included A B C We previously reported that replacing Ser13 in Cdc37 with a nonphosphorylatable amino acid abolished the binding of Cdc37 to Hsp90 client signaling protein kinases [26,27] Using the phospho-specific antibody Fig Substrate specificity of the antibody against CK2-phosphorylated Cdc37 (A) Alignment of the amino acid sequences surrounding the CK2 phosphorylation sites of Cdc37 (rat), FKBK52 (rabbit), and Hsp90 (human) The position of the CK2-catalyzed phosphorylation sites is indicated by an arrow (B) Recombinant purified Cdc37(WT) (lanes and 5), Cdc37(13SA) (lanes and 6), FKBP52 (lanes and 7) or Hsp90 (lanes and 8) was incubated alone (lanes 1–4) or with purified CK2 (lanes 5–8) in the presence of Mg2+-[32P]ATP[cP], and phosphorylated proteins were visualized by autoradiography after SDS ⁄ PAGE (C) The same protein mixtures as shown in (B) were analyzed by western blotting with anti-[pSer13]Cdc37 (D) The same protein mixtures shown in (B) and (C) were stained with CBB after SDS ⁄ PAGE The positions of molecular weight markers, as well as Hsp90, FKBP52, and Cdc37, are shown D FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5693 Cdc37 phosphorylation by CK2 and signaling kinases Y Miyata and E Nishida The amounts of Hsp90, total Cdc37, CK2-phosphorylated Cdc37 and immunoprecipitated kinases were examined by western blot analysis, using the corresponding antibodies As shown in Fig 3, each kinase, Cdk4 (lane 4), MOK (lane 5), v-Src (lane 6), and Raf1 (lane 7), associated specifically with Hsp90 (Fig 3A) and with Cdc37 (Fig 3B) Importantly, anti-[pSer13]Cdc37 recognized Cdc37 in all Hsp90–client protein kinase complexes (Fig 3C), indicating that CK2-phosphorylated Cdc37 was present in these kinase complexes The control kinases, CK1 (lane 2) and DYRK2 (lane 3), did not bind to Hsp90 (Fig 3A), Cdc37 (Fig 3B), or phospho-Cdc37 (Fig 3C), although comparable amounts of protein were immunoprecipitated for each kinase, as shown on western blots obtained using anti-FLAG (Fig 3D) Intracellular distribution of CK2-phosphorylated Cdc37 As shown above, CK2-phosphorylated Cdc37 associates with signaling protein kinases, so intracellular regions where signaling protein kinases accumulate might also be expected to contain high concentrations of phosphorylated Cdc37 Growth factors such as epidermal growth factor (EGF) and insulin-like growth factor-I are known to induce membrane ruffling, and actin cytoskeleton and signaling molecules, such as Rho family G-proteins and protein kinases, are known to accumulate in these areas We therefore examined the intracellular distribution of CK2-phosphorylated Cdc37 in EGF-stimulated KB cells, a cell line known to show prominent membrane ruffling in response to growth factors [34] KB cells were serum starved by incubating them in medium containing only 1% fetal bovine serum for h and then incubated with or without 30 nm EGF for Anti-Hsp90, anti-Cdc37 and anti-[pSer13]-Cdc37 were used to immunofluorescently stain the cells, to examine the intracellular distributions of the proteins Before EGF stimulation, Hsp90 was mainly localized in the cytoplasm, whereas Cdc37 and phospho-Cdc37 were present throughout the KB cells, both in the cytoplasm and in the nucleus (Fig 4A–C) EGF induced membrane ruffling and Hsp90 was localized to the membrane ruffles (Fig 4D, arrowheads) as previously reported [35] Cdc37 also accumulated in areas of membrane ruffling in EGFstimulated KB cells (Fig 4E, arrowheads), as did CK2-phosphorylated Cdc37 (Fig 4F, arrowheads) These results indicated that phosphorylated Cdc37 colocalized with Hsp90 in the growth factor-induced membrane ruffles, where signaling protein kinases also accumulated, and are consistent with the results in 5694 A B C D Fig Association of Ser13-phosphorylated Cdc37 with various Hsp90 client protein kinases COS7 cells were transfected with empty vector DNA (lane 1, control), or plasmids encoding FLAGtagged protein kinases CK1 (lane 2, control), DYRK2 (lane 3, control), Cdk4 (lane 4), MOK (lane 5), v-Src (lane 6), or Raf1 (lane 7), and the kinase–chaperone complexes were immunopurified The amounts of Hsp90 (A), Cdc37 (B), phosphorylated Cdc37 (C) and protein kinase (D) in the kinase–chaperone complexes were assessed by western blotting with anti-Hsp90, anti-Cdc37, anti[pSer13]-Cdc37, and anti-FLAG, respectively The positions of molecular weight markers are shown Fig 3, showing that phosphorylated Cdc37 forms complexes with Hsp90 client signaling protein kinases We next investigated whether the accumulation of phosphorylated Cdc37 in the EGF-induced membrane ruffles was a result of an increase in the phosphorylation of Cdc37 by activated CK2 Serum-starved KB cells were treated with EGF for up to 60 min, and the levels of phosphorylated Cdc37 in total cell extracts were determined by western blotting using FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida Cdc37 phosphorylation by CK2 and signaling kinases A B C D E F Fig Subcellular localization of phosphorylated Cdc37 in growth factor-induced membrane ruffles KB cells incubated in low-strength serum (1%) were untreated (A–C) or treated with 30 nM EGF for (D–F) The intracellular localization of Hsp90 (A, D), total Cdc37 (B, E) or the phosphorylated form of Cdc37 (C, F) are shown in the left columns by immunofluorescent microscopy Corresponding phase contrast images are shown in the right columns For each panel, two typical images (top and bottom) from different fields are shown anti-[pSer13]-Cdc37 (Fig 5A) The levels of total Cdc37 (Fig 5B), phosphorylated extracellular signalregulated kinase (ERK) (Fig 5C) and total ERK (Fig 5D) were also measured by western blotting using anti-Cdc37, anti-[pTEpY]-ERK (an antibody specific for the dually phosphorylated, activated form of ERK), and anti-ERK, respectively The results clearly showed that the levels of phosphorylated Cdc37 as well as total Cdc37 did not change after EGF stimulation (Fig 5A,B) The activation of EGF-induced signaling pathways under these conditions was confirmed by the observation that EGF rapidly stimulated the dual phosphorylation of ERK (Fig 5C) These results agree with an earlier report showing that growth factors did not significantly activate CK2 in cells [36] In addition, we noted that anti-[pSer13]-Cdc37 recognized a single protein band in western blots of whole cell lysates (Fig 5A), reinforcing our conclusion that anti[pSer13]-Cdc37 specifically recognizes phospho-Cdc37 but not other CK2 substrates (Fig 2) Taking these findings together, we concluded that CK2-phosphorylated Cdc37 accumulates in EGF-induced membrane ruffles as a result of intracellular redistribution of phospho-Cdc37 rather than a net increase in the phosphorylation of Cdc37 by CK2 within the cell Analysis of Cdc37 phosphorylation by phospho-affinity gel electrophoresis We wanted to establish a simple biochemical method for analyzing the phosphorylation of Cdc37 To this end, we investigated separating phosphorylated Cdc37 from nonphosphorylated Cdc37 by phospho-affinity gel electrophoresis, a technique recently developed by Kinoshita et al [5] Purified recombinant Cdc37 was incubated with CK2a or CK2 holoenzyme or alone, in the presence or absence of ATP and ⁄ or Mg2+, and the mixtures were analyzed by phospho-affinity gel electrophoresis CBB staining of the phospho-affinity gel showed that the mobility of Cdc37 was markedly decreased when it had been incubated with CK2a or CK2 holoenzyme in the presence of both ATP and Mg2+ (Fig 6A, lanes and 9) This mobility shift was not a nonspecific effect due to ATP and ⁄ or Mg2+, because no shift was observed in the absence of CK2 (Fig 6A, lanes 1–3) Nor was the mobility shift caused by the physical association of CK2 and Cdc37, as CK2 did not induce the Cdc37 mobility shift in the absence of ATP or Mg2+ (Fig 6A, lanes 4, 5, and 8) The CK2a- or CK2 holoenzyme-dependent phosphorylation of Cdc37 in the presence of Mg2+–ATP FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5695 A 60 30 15 EGF Cdc37 phosphorylation by CK2 and signaling kinases Y Miyata and E Nishida 62 47 33 WB: Anti-[pSer13]-Cdc37 B 62 47 33 WB: Anti-Cdc37 C 62 47 33 WB: Anti-[pTEpY]-ERK D 62 47 33 WB: Anti-ERK Fig Effect of EGF treatment on the phosphorylation of Cdc37 KB cells incubated in low-strength serum (1%) were untreated (lane 1) or treated with 30 nM EGF for (lane 2), 15 (lane 3), 30 (lane 4), or 60 (lane 5) Cell extracts were prepared, and phosphorylated Cdc37 (A), total Cdc37 (B), dually phosphorylated and activated ERK (C) and total ERK (D) were labeled on western blots with the corresponding antibodies was confirmed by labeling with anti-[pSer13]-Cdc37 (Fig 1A) It should be noted that no band shift was detected when the protein mixtures were analyzed by normal SDS ⁄ PAGE (Fig 1B,D) We then examined the effect of Cdc37 mutations at the CK2 phosphorylation site on phospho-affinity gel electrophoresis mobility Recombinant purified wildtype Cdc37 protein or Ser13 mutants of Cdc37, 5696 Cdc37(13SA) and Cdc37(13SD) were incubated with CK2a or CK2 holoenzyme in the presence of Mg2+–ATP and analyzed by phospho-affinity gel electrophoresis In contrast to the CK2a- or CK2 holoenzyme-dependent mobility shift shown by wildtype Cdc37 (Fig 6B, lanes and 7), the mobilities of Cdc37(13SA) and Cdc37(13SD) were not affected by CK2a or CK2 (Fig 6B, lanes 5, 6, and 9), indicating that the mobility shift of Cdc37 in phospho-affinity gel electrophoresis was caused by its CK2-dependent phosphorylation on Ser13 In addition, these results showed that CK2a phosphorylated Cdc37 only on Ser13, as in the case of CK2 holoenzyme, even in the absence of CK2b We therefore used CK2a to phosphorylate Cdc37 in vitro in subsequent experiments Phospho-affinity gel electrophoresis can be combined with other detection systems, including autoradiography and western blotting Cdc37 was incubated with or without CK2a in the presence of Mg2+–ATP, and separated by phospho-affinity gels The phospho-affinity gels were washed with a buffer containing EDTA to remove Mn2+, and thereby remove the phosphatebinding activity of Phos-tag, so that the phosphoproteins in the gel could be transferred to a membrane The membrane was probed with anti-Cdc37 or anti[pSer13]-Cdc37 Anti-Cdc37 labeled both the lowmobility and high-mobility Cdc37 bands (Fig 7A, lanes and 2), whereas anti-[pSer13]-Cdc37 recognized only the low-mobility Cdc37 band (Fig 7B, lane 2), indicating that the band with the decreased mobility represented Cdc37 in the Ser13-phosphorylated form When we carried out the phosphorylation reaction in the presence of [32P]ATP[cP], analyzed the mixtures by phospho-affinity gel electrophoresis and autoradiographed the gels, radioactivity was detected only in the low-mobility band using wild-type Cdc37 (Fig 7C, lane 3) Neither the mobility shift nor the radioactivity could be detected when CK2a was omitted (Fig 7C,D, lane 2) or when the phosphorylation site mutant Cdc37(13SA) was used (Fig 7C,D, lane 4) We therefore concluded that CK2 phosphorylates only one residue, Ser13, in Cdc37, and that this single phosphorylation causes the Cdc37 mobility shift seen in phospho-affinity gel electrophoresis Analysis of Cdc37 phosphorylation and dephosphorylation by phospho-affinity gel electrophoresis To analyze the time course of Cdc37 phosphorylation in vitro, mixtures of recombinant Cdc37 and CK2a, in the presence of Mg2+–ATP, were sampled at different time points, and the samples were analyzed by FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida A B Fig Analysis of Cdc37 phosphorylation by phospho-affinity gel electrophoresis (A) Recombinant Cdc37 alone (lanes 1–3), with CK2a (lanes 4–6) or with CK2 holoenzyme (lanes 7–9), in the presence (+) or absence (–) of Mg2+ and ⁄ or ATP as indicated, was incubated at 30 °C for 30 min, analyzed by phospho-affinity gel electrophoresis, and then stained with CBB (B) Wild-type protein (lanes 1, and 7) and two Cdc37 mutant proteins, 13SA (lanes 2, and 8) and 13SD (lanes 3, and 9), were incubated alone (lanes 1–3), with CK2a (lanes 4–6) or with CK2 holoenzyme (lanes 7–9) in the presence of Mg2+–ATP for 30 at 30 °C The phosphorylation mixtures were analyzed by phospho-affinity gel electrophoresis and stained with CBB phospho-affinity gel electrophoresis Phosphorylation was rapid and detected within (Fig 8A, lane 4) and completed within at 30 °C (Fig 8A, Cdc37 phosphorylation by CK2 and signaling kinases lanes 4–7) The transition in mobility from the higher to lower bands was direct, with no bands of intermediate mobility being detected, supporting our previous conclusion that CK2a phosphorylates only one site in Cdc37 We then studied the time course of dephosphorylation by incubating CK2a-phosphorylated Cdc37 (Fig 8B, lane 2) with (Fig 8B, lanes 7–10) or without (Fig 8B, lanes 3–6) k-phosphatase in the presence of the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and analyzing the products by phospho-affinity gel electrophoresis and western blotting using antiCdc37 Incubating phospho-Cdc37 with k-phosphatase induced rapid dephosphorylation, with a high-mobility Cdc37 band appearing within (Fig 8B, lane 8) Again, the band shift induced was direct, from the low-mobility to high-mobility bands, with no intermediate bands being detected Analysis of signaling kinase–Hsp90–Cdc37 complexes by phospho-affinity gel electrophoresis Finally, we analyzed complexes between signaling protein kinases and Hsp90–Cdc37 molecular chaperones by phospho-affinity gel electrophoresis The Hsp90 client kinases Cdk4, MOK, v-Src and Raf1 were expressed as FLAG-tagged fusion proteins in COS7 cells, and the kinase–chaperone complexes were immunopurified using anti-FLAG agarose The immunocomplexes were separated by phospho-affinity gel electrophoresis, transferred to western blots, and labeled with anti-Cdc37 or anti-[pSer13]-Cdc37 Specific associations between Cdc37 and Cdk4, MOK, v-Src and Raf1 were observed (Fig 9A), consistent with the result shown in Fig Interestingly, Cdc37 in the protein kinase complexes was detected as a single band in phospho-affinity gels (Fig 9A), and the band was recognized by anti-[pSer13]-Cdc37 (Fig 9B), indicating for the first time that all of the Cdc37 in the signaling kinase–Hsp90 complexes was in its Ser13phosphorylated form in vivo Discussion In this study, we have produced a phospho-specific antibody against Cdc37, which recognized recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg2+ and ATP The specificity of this antibody was demonstrated by showing that it did not recognize mutant Cdc37 in which the CK2 phosphorylation site, Ser13, had been replaced with nonphosphorylatable amino acids, and it did not recognize other CK2-phosphorylated proteins such as Hsp90 and FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5697 Cdc37 phosphorylation by CK2 and signaling kinases A Y Miyata and E Nishida B C D Fig Characterization of Cdc37 phosphorylation by phospho-affinity gel electrophoresis Recombinant Cdc37 was phosphorylated with CK2a and analyzed by phospho-affinity gel electrophoresis (A) Non-phosphorylated (lane 1) and phosphorylated (lane 2) Cdc37 were separated by phospho-affinity gel electrophoresis and analyzed by western blotting with anti-Cdc37, for total Cdc37 (B) The same membrane as in (A) was stripped and reprobed with anti-[pSer13]-Cdc37 (C) Wild-type protein [Cdc37(WT)] (lane 3) and a mutant [Cdc37(13SA)] (lane 4) of Cdc37 were phosphorylated with CK2a in the presence of [32P]ATP[cP] and analyzed by phospho-affinity gel electrophoresis followed by autoradiography As controls, CK2a alone (lane 1) or Cdc37(WT) alone (lane 2) was incubated under the same conditions (D) CBB staining of the same gel as in (C) FKBP52 Thus, the antibody specifically recognizes Cdc37 only when it is phosphorylated on Ser13 by CK2 Using this antibody, we have shown that complexes between Hsp90 and its client signaling kinases Cdk4, MOK, v-Src and Raf1 contain CK2-phosphorylated Cdc37 in vivo These results are consistent with previous reports that CK2 phosphorylates Cdc37 on Ser13 both in vivo and in vitro, and that this phosphorylation is essential for the binding of Cdc37 to multiple Hsp90 client protein kinases [25,26,28] Immunofluorescence staining with the antibody showed that phosphorylated Cdc37 accumulated in growth factor-induced membrane ruffles in KB cells This accumulation was the result of a redistribution of phospho-Cdc37 within the cells rather than an upregulation of total Cdc37 phosphorylation by CK2 after EGF stimulation Previously, Hsp90 has been reported to bind to polymerized actin and to accumulate in growth factor-induced membrane ruffles, where actin filaments are abundant [35] In fact, Hsp90 has been suggested to be involved in the intracellular distribution and trafficking of many signaling molecules by interacting with its client proteins and the cytoskeletal architecture [37,38] Membrane ruffling is one of the morphological responses rapidly induced in cells by growth factor stimulation, and the accumulation of a 5698 variety of signaling molecules, including receptor tyrosine kinases and G-proteins, in membrane ruffles has been reported [39] This might be why Hsp90 and phosphorylated Cdc37 accumulate in membrane ruffles, as only the phosphorylated form of Cdc37 is active in recruiting Hsp90 to its client signaling kinases [26] The Hsp90–Cdc37–kinase complexes might then interact with the actin cytoskeleton via Hsp90 in the membrane ruffling region Membrane ruffling has been related to the metastatic status of tumor cells, and it has been suggested as an indicator of tumor cell motility and metastatic potential [40] Therefore, the inhibition of Hsp90, Cdc37 or CK2 might influence, not only signaling affecting cell growth, but also the metastasis of neoplastic cells In this study, we exploited the recently developed compound Phos-tag, which has specific phosphatebinding activity [4,5], to separate phosphorylated and nonphosphorylated forms of Cdc37 by phospho-affinity gel electrophoresis We have demonstrated a specific mobility shift in Cdc37 only when it is incubated with CK2 in the presence of Mg2+ and ATP Replacing the CK2 phosphorylation site in Cdc37 with nonphosphorylatable amino acids completely abolished this mobility shift The low-mobility Cdc37 band was both the only band recognized by the phospho-specific FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida A B Fig Time course of Cdc37 phosphorylation and dephosphorylation analyzed by phospho-affinity gel electrophoresis (A) Time course of CK2-dependent Cdc37 phosphorylation was analyzed by phospho-affinity gel electrophoresis, between (lane 1) and 10 (lane 9) (B) Time course of the dephosphorylation of CK2phosphorylated Cdc37 by k-phosphatase was analyzed by phosphoaffinity gel electrophoresis Lane 1, nonphosphorylated Cdc37; lane 2, Cdc37 phosphorylated with CK2a Phosphorylated Cdc37, as in lane 2, was incubated with (lanes 7–10) or without (lanes 3–6) k-phosphatase in the presence of the CK2 inhibitor TBB for up to 20 The mixtures were analyzed by phospho-affinity gel electrophoresis followed by western blotting with anti-Cdc37 Samples were analyzed after dephosphorylation for (lanes and 7), (lanes and 8), 10 (lanes and 9), and 20 (lanes and 10) antibody against Cdc37 and the only band that became radioactive after incubation with CK2 in the presence of [32P]ATP[cP] Using this new technique, we have been able to strengthen our previous conclusion that CK2 rapidly phosphorylates Cdc37 at only one site, Ser13, and to demonstrate for the first time that only phosphorylated Cdc37 is present in signaling protein kinase complexes with Hsp90 These results further support the previous proposal that the phosphorylation of Cdc37 by CK2 is essential for its protein kinase-binding activity [25,26,28] Future studies will be needed to elucidate whether the phosphorylation of Cdc37 fluctuates dynamically within cells according to cellular conditions Cdc37 phosphorylation by CK2 and signaling kinases Phospho-affinity gel electrophoresis is a simple and easy method with the advantage that it uses no radioactive material Moreover, it is a technique that could be used for the analysis of virtually any phosphoprotein, as a phosphorylation-dependent mobility shift in these gels could occur for any protein It should be noted, however, that large excesses of metal chelators such as EDTA and chemicals containing phosphate moieties, including sodium phosphate, b-glycerophosphate or ATP, may interfere with the assay In addition, crude protein mixtures such as tissue extracts that may contain a large amount of phosphoproteins may not be easy to analyze directly with this technique Further improvements in phospho-affinity gel electrophoresis might be possible and might broaden its applicability Previous studies have identified many substrates for CK2 [21], and demonstrated its involvement in many different cellular functions, including cell division, cell survival, and gene expression [19,20,24] Surprisingly, however, the regulatory mechanism of CK2 in cells remains largely unknown Observations that CK2 activity was enhanced by growth factors have been challenged [36], and this discrepancy arose partly from the lack of a reliable method for quantifying CK2 activity in vivo Our results agree with those of previous studies [25,26,28] in demonstrating that CK2 phosphorylates only one site in Cdc37, so the phosphorylation state of Cdc37 should be an index of CK2 activity both in vivo and in vitro The phosphorylation of Cdc37 can now be readily monitored using the phospho-specific antibody and phospho-affinity gel electrophoresis, as described here Several lines of evidence suggest a critical role for CK2 in tumorigenesis [22] For example, CK2 is tumorigenic when overexpressed in a transgenic mouse [41] As the CK2–Cdc37 module has been suggested to function as a master switch with a positive feedback mechanism for various signaling protein kinases [25,31], the development of a simple method for determining CK2 activity and Cdc37 phosphorylation is likely to be both biologically and clinically important in the future Experimental procedures Plasmids, proteins and antibodies Plasmids that encode the FLAG-tagged protein kinases Cdk4, MOK, v-Src and Raf1 for expression in mammalian cells have been described previously [26] cDNA for Zebrafish CK1a was a kind gift from J E Allende (Universidad de Chile), and an EcoRI fragment of the coding region was inserted into an EcoRI site of pCMV–Tag2B to obtain an expression plasmid encoding FLAG-tagged CK1 An expression plasmid for FLAG-tagged DYRK2 used as a FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5699 Cdc37 phosphorylation by CK2 and signaling kinases A Y Miyata and E Nishida B Fig Analysis of the signaling protein kinase–chaperone complexes by phosphoaffinity gel electrophoresis Hsp90 client kinases Cdk4 (lane 2), MOK (lane 3), v-Src (lane 4) and Raf1 (lane 5) were expressed in COS7 cells and immunopurified The protein kinase complexes were analyzed by phospho-affinity gel electrophoresis and western blotting with anti-Cdc37 (A) or anti-[pSer13]Cdc37 (B) COS7 cells transfected with DNA from the empty vector were used as a control (lane 1) The position of Cdc37 in the gel is shown on the right control will be described elsewhere A dual expression plasmid (CK2a + Cdc37 ⁄ pETDuet-1b) that encodes His6– CK2a and Cdc37 has been described previously [27] A BamHI fragment encoding full-length human Hsp90a, described previously [27], was inserted into a BamHI site in pGEX6P2 to obtain a bacterial expression plasmid for human Hsp90a Wild-type [Cdc37(WT)] and mutant [Cdc37(13SA) and Cdc37(13SD)] recombinant rat Cdc37 proteins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified as described previously [26] Native CK2 holoenzyme (a2b2) was purified from porcine testes as described previously [30] The expression and purification of recombinant FKBP52 has been described previously [32] EGF was purchased from BD Biosciences (San Jose, CA, USA) and k-phosphatase from Upstate Biotechnologies (Lake Placid, NY, USA) Horseradish peroxidase (HRP)-conjugated anti-Cdc37 (E-4) was purchased from Santa Cruz (Santa Cruz, CA, USA), anti-FLAG (M2) and anti-FLAG (M2) affinity gel from Sigma (Saint Louis, MO, USA), anti-ERK2 (clone D-2) from Santa Cruz, and antibody specific for the dually phosphorylated (pTEpY) ERK (V803A) from Promega (Madison, WI, USA) Anti-Hsp90 has been described previously [35] Antibody specific for phosphorylated Cdc37 was raised in rabbits against a phospho-peptide, VWDHIEVpSDDEDETHC, corresponding to amino acids 6–20 of Cdc37 with an additional C-terminal cysteine for conjugation Rabbit antisera were affinity purified using the phosphopeptide, and then immunoadsorbed with an affinity resin conjugated with the nonphosphorylated form of the peptide to remove antibodies reactive to this nonphosphorylated form The peptides were synthesized by MBL Co (Nagoya, Japan), and immunizations and affinity purification were performed at SCRUM Inc (Tokyo, Japan) 5700 Expression and purification of Hsp90–client protein kinase complexes COS7 cells were cultured and transfected with mammalian expression vectors by electroporation, and cell extracts were prepared in an extraction buffer (50 mm Tris ⁄ HCl, 10% glycerol, 100 mm NaF, 50 mm NaCl, mm EDTA, mm sodium orthovanadate, 10 mm sodium pyrophosphate, mm dithiothreitol, 1% Nonidet P-40, pH 8.0) as described previously [32] Extracts containing equal amounts of protein were incubated with 40 lL of anti-FLAG affinity gel for 12 h at °C The immunocomplexes were extensively washed with the extraction buffer, and then shaken with 0.33 mgỈmL)1 (final concentration) · FLAG peptide (Sigma) in 50 lL of extraction buffer at °C for h Proteins eluted from anti-FLAG affinity gel were collected by brief centrifugation (13 000 g for at °C using an Eppendorf 5415R centrifuge and wide angle rotor), and then treated in Amicon 0.22 lm Ultrafree centrifugal filtration tubes (Millipore, Bedford, MA, USA) For phospho-affinity gel electrophoresis analysis, Tris-buffered saline (50 mm Tris ⁄ HCl, 150 mm NaCl, pH 7.4) was used for washing and elution instead of extraction buffer Expression and purification of CK2a Introduction of the dual expression plasmid CK2a + Cdc37 ⁄ pETDuet-1b into the host E coli BL21 CodonPlus (DE3) RIL (Stratagene, La Jolla, CA, USA) and induction of the expression of CK2a have been described previously [27] E coli cell pellets were collected from 500 mL cultures and solubilized in 35 mL of B-PER bacterial extraction solution (Pierce, Rockford, FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Miyata and E Nishida IL, USA) supplemented : 100 (v ⁄ v) with an EDTA-free protease inhibitor cocktail (Sigma) After the addition of m NaCl to adjust the final concentration of NaCl to 700 mm, the mixture was gently agitated for 20 at °C using an Avanti HP-25 The mixture was centrifuged at 18 000 g for 15 at °C, and the supernatant was filtered through a MillexHV 0.45 lm filter unit (Millipore) His6-tagged CK2a was purified on a 10 mL column of Talon beads (Clontech-Takara BIO, Mountain View, CA, USA) with a linear gradient of 0–500 mm imidazole in 50 mm sodium phosphate and 300 mm NaCl (pH 7.0) Fractions containing CK2a were concentrated using an Amicon Ultra-15 Centrifugal Filter Device (Millipore) CK2a was further purified on a Superdex 200 HR10 ⁄ 30 gel filtration column (GE Healthcare Biosciences, Uppsala, Sweden) with a buffer containing 50 mm Tris, mm EDTA, mm dithiothreitol, and 200 mm NaCl (pH 7.4) Fractions containing CK2a were further purified using a ResourceQ column (GE Healthcare Biosciences) and a linear gradient of 0–1000 mm NaCl in 50 mm Tris, mm EDTA, and mm dithiothreitol (pH 9.0) Purified CK2a was dialyzed in the same buffer containing 200 mm NaCl This recombinant CK2a was more than 98% pure and ran as a single band on SDS ⁄ PAGE stained with CBB Expression and purification of human Hsp90a The bacterial expression plasmid encoding GST-tagged human Hsp90a (Hsp90a ⁄ pGEX6P2) was introduced into a host E coli strain BL21 CodonPlus (DE3) RIL After reaching an A600 nm level of 0.7, 0.1 mm (final concentration) isopropyl-1-thio-b-d-galactopyranoside was added to induce expression, and the culture was shaken for h at 30 °C A frozen E coli cell pellet from a 500 mL culture was solubilized in 40 mL of B-PER solution (Pierce) supplemented : 100 (v ⁄ v) with a bacterial protease inhibitor cocktail (Sigma) Purification of GST–Hsp90a and cleavage with PreScission Protease (GE Healthcare Biosciences) to remove the GST moiety from the fusion protein were performed essentially as described previously [11] Hsp90a was further purified by ResourceQ column chromatography using a linear gradient of 0–1000 mm NaCl in 50 mm Tris ⁄ HCl, mm EDTA, and mm dithiothreitol (pH 7.4) This recombinant Hsp90a was more than 95% pure and ran as a single band on SDS ⁄ PAGE stained with CBB Phosphorylation with CK2 and dephosphorylation with k-phosphatase Recombinant purified Cdc37, FKBP52 or Hsp90 were shaken with purified native CK2 holoenzyme or with recombinant purified CK2a at 30 °C for 30 (or for the indicated time periods) The standard phosphorylation Cdc37 phosphorylation by CK2 and signaling kinases mixture was 85 mm Tris, mm Hepes, 100 mm NaCl, 10 mm MgCl2, 0.2 mm EDTA, 0.2 mm dithiothreitol, 7.5 mm ATP (or 0.75 mm ATP including [32P]ATP[cP] for radioactive assays), 0.5 mgỈmL)1 substrate protein, and 0.096 mgỈmL)1 CK2 holoenzyme or CK2a (pH 7.5) In the experiments shown in Figs and 8, the phosphorylation mixture contained 265 mm NaCl Native CK2, CK2a, ATP and ⁄ or MgCl2 were omitted from the incubation mixtures where indicated For dephosphorylation, k-phosphatase was added and incubated for the indicated time periods at 37 °C in the presence of a specific CK2 inhibitor, TBB The final incubation buffer for phosphatase treatment was 70 mm Tris, 57 mm Hepes, 220 mm NaCl, mm MgCl2, mm MnCl2, 0.26 mm EDTA, mm dithiothreitol, 6.1 mm ATP, 20 lm TBB, 2% dimethylsulfoxide, 0.4 mgỈmL)1 Cdc37, 0.08 mgỈmL)1 CK2a, and 0.4 unitỈlL)1 k-phosphatase Phosphorylation and dephosphorylation reactions were stopped by adding : (v ⁄ v) SDS sample buffer followed by incubation at 98 °C for Immunofluorescent staining KB cells were cultured on glass coverslips in DMEM supplemented with 10% fetal bovine serum To serum starve cells, the medium was replaced with DMEM + 1% fetal bovine serum for h EGF was added to cultures at a final concentration of 30 nm, and cells were incubated at 37 °C for to induce membrane ruffling Cells were fixed with formaldehyde, permeabilized, and stained with antibodies as described previously [27] Phospho-affinity polyacrylamide gel electrophoresis Phos-tag acrylamide was purchased from the Phos-Tag Consortium (http://www.phos-tag.com), and mm stock solutions in water were prepared Phospho-affinity gel electrophoresis was performed essentially as described previously [5] The final composition of phospho-affinity SDS-polyacrylamide gels was 7.8% acrylamide, 0.21% bisacrylamide, 375 mm Tris ⁄ HCl, pH 8.8, 0.1% SDS, 0.05% ammonium persulfate, 0.086% TEMED containing 50 lm Phos-tag acrylamide, and 100 lm MnCl2 The stacking gel solution used was 3.9% acrylamide, 0.11% bis-acrylamide, 124 mm Tris ⁄ HCl, pH 6.8, 0.1% SDS, 0.1% ammonium persulfate, and 0.1% TEMED The electrophoresis running buffer used was 25 mm Tris, 192 mm glycine, and 0.1% SDS (pH 8.3) Heat denaturation with SDS, sample loading and CBB staining procedures were the same as for ordinary SDS ⁄ PAGE The final concentration of NaCl in the samples was adjusted to 265 mm before heat denaturation Electrophoresis was stopped when a prestained 40 kDa protein marker reached the front edge of the gels, to give clear band separation FEBS Journal 274 (2007) 5690–5703 ª 2007 The Authors Journal compilation ª 2007 FEBS 5701 Cdc37 phosphorylation by CK2 and signaling kinases Y Miyata and E Nishida Other procedures Normal SDS ⁄ PAGE was performed with 10% acrylamide gels Western blotting was performed with HRP-conjugated secondary antibodies or peroxidase-conjugated antibodies and a chemiluminescent detection system as described previously [26] For western blotting after phospho-affinity gel electrophoresis, gels were 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Koyasu S, Nishida E, Kadowaki T, Matsuzaki F, Iida K, Harada F, Kasuga M, Sakai H & Yahara I (1986) Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins Proc Natl Acad