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Mol. Cells, Vol. 25, No. 1, pp. 43-49 Molecules and Cells ©KSMCB 2008 Proteomics Analysis of Immunoprecipitated Proteins Associated with the Oncogenic Kinase Cot Binhui Wu and Rupert C. Wilmouth* School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore. (Received April 9, 2007; Accepted August 9, 2007) Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to proinflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: Cot130-399 (kinase domain), Cot1-388 (N-terminal and kinase domains), Cot1-413, Cot1-438 (containing a putative PEST sequence), Cot1-457 (containing both PEST and degron sequences) and Cot1-467 (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silverstained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immunoprecipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells. Keywords: Chaperone; Cot; Hsp90; MAP3K. Introduction The human Cancer Osaka thyroid (Cot) gene encodes a mitogen-activated protein kinase kinase kinase (MAP3K) which when overexpressed has been shown to activate the MAP kinases ERK, JNK and p38 (Chiariello et al., 2000; Salmerón et al., 1996). These pathways link Cot activity with several transcription factors, including NF-κB, NFAT and AP-1 (Lin et al., 1999; Tsatsanis et al., 1998). However, in experiments using tpl2-/- knockout mice, it * To whom correspondence should be addressed. Tel: 65-6316-2845; Fax: 65-6791-3856 E-mail: [email protected] was shown that Tpl-2, the rat homologue of Cot, was required only for ERK and not JNK or p38 activation in LPS-stimulated macrophages (Dumitru et al., 2000). The oncogenic form of Cot contains a C-terminal truncation where the last 69 amino acids of the wild-type protein are replaced with an unrelated 18 amino acid sequence (Aoki et al., 1993). Similarly, disruption of the tpl-2 gene by insertion of the Moloney leukemia virus leads to the expression of a truncated protein, where the final 44 amino acids of the wild-type protein are replaced with an unrelated 10 amino acid sequence, and unmasking of its oncogenic potential (Makris et al., 1993). The Tpl-2 locus has been shown to be involved in the induction of mammary carcinomas in mice and may be a key molecule for the study of human breast cancer (Erny et al., 1996; Sourvinos et al., 1999). Full-length Cot protein contains 467 residues and three domains. The N-terminal domain, whose precise function remains unclear, consists of residues 1 to 132. The region from 133 to 388 is the kinase domain which has signifycant sequence similarity with kinase domains from other MAP3Ks. The putative C-terminal domain consists of the residues 389 to 467. It has been shown that C-terminally truncated Tpl-2 (oncogenic form) is approximately five times more active in autophosphorylation and five and seven times more active in the phosphorylation of histone substrates H3 and H2A, respectively (Ceci et al., 1997). In the same study, it was suggested that the C-terminal tail might fold onto the kinase domain and hence inhibit its activity. Tpl-2 transgenic mice expressing the wild-type protein did not exhibit a biological phenotype, whereas those expressing the truncated protein developed largeAbbreviations: AP-1, activating protein-1; ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; NFAT, nuclear factor of activated T cells; TNF-α, tumour necrosis factor-α. 44 Proteomics Analysis of Proteins Associated with Cot cell lymphoblastic lymphomas of T-cell origin. It was shown that C-terminally truncated Cot kinase (oncogenic form) has a longer half-life (95 min) than the wild-type protein (35 min) (Gándara et al., 2003). Addition of a proteasome inhibitor to total cell extracts reduced degradation. After 30 min incubation with the 20S proteasome, 55% of wild-type Cot was degraded compared to 20% of truncated Cot. It was found that the region encompassing residues 435-457 in the C-terminal domain of Cot (termed a ‘degron’) targeted the protein for proteasome-mediated degradation. The truncated Cot appeared to have higher specific activity than the wild-type protein, although the level of phosphorylation of the substrate ERK was similar. The region between residues 425 and 467 (termed a ‘kinase repression domain’) was thought to be largely responsible for the difference in activity. A putative PEST sequence was also found between residues 415 and 438. In this study, we attempted to determine potential binding partners to Cot and their region of association by separating proteins that were pulled-down with various Cot constructs following immunoprecipiation. The separation of the proteins was carried out using 2D electrophoresis, which is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells and tissues. Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Each spot can then be excised from the gel and the protein contained within extracted. The identity of the protein can be ascertained by trypsin digestion followed by mass spectrometric analysis of the resultant peptides. This procedure of separating and analysing a complex mixture of proteins is generally known as proteomics (Vercauteren et al., 2006). Materials and Methods Cloning and cell culture The Cot gene was purchased from Genecopeia (USA). N-terminal 6xHis-tagged Cot130-399, Cot1-388, Cot1-413, Cot1-438, Cot1-457, Cot1-467 were constructed by subcloning EcoRI- and XhoI-digested PCR fragments into pcDNA4 HisMax (Invitrogen). HEK293 cells were used as transfection hosts, and were cultured in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), L-glutamine (4 mM), sodium pyruvate (1 mM), ampicillin (100 μg ml−1) and streptomycin (100 μg ml−1) at 37 °C in a humidified atmosphere of 5% CO2 . The cells were grown in 100 mm dishes or 96-well plates and were transfected transiently at 60−80% confluency by using a standard calcium phosphate method with 10 μg constructs. Cells were harvested at 48 hr after transfection for the expression test and 2D gel analysis, or changed to medium without fetal calf serum for overnight starvation. Cells were left untreated or treated with LPS (200 ng ml−1) or TNF-α (20 ng ml−1) for another 6 h for the in vivo kinase activity analysis. For the experiments on endogenous Cot, Hela cells were cultured in an identical fashion to the HEK293 cells. Western blotting and immunoprecipitation Anti-6xHis (H20) antibody was from Delta Biolabs; anti-Cot (N17), anti-Cot (M20), anti-Cot (H212), anti-PDI (H160), anti-p105/p50 (E10), anti-phospho-MEK1/2 (S217/221), anti-MEK1/2 (H8), antiHsp90α/β (F8), anti-Hsp70 (W27), anti-Grp78 (C20), antiAkt1/2 (H136) and anti-Abin2 (N16) antibodies were from Santa Cruz. Cells were lysed in buffer (25 mM TRIS pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 20 mM β-glycerophosphate, 1 mM Na3VO4, Roche Complete protease inhibitor) and the soluble fraction obtained by centrifugation (14,500 × g, 10 min) at 4°C. The total protein concentration was determined by the Bradford assay. For Western blotting, 50 μg of total protein was separated by SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare) by semi-dry blotting. The membrane was blocked for 1 h with 2% BSA in TBS-T (triethanolamine-buffered saline with 0.1% Tween) and incubated for 1 hr with primary antibody. After washing (3 times) with TBS-T, the membrane was incubated for another 1 h with secondary antibody. After washing (3 times) with TBS-T, the membrane was visualised by chemiluminescence (ECL plus, GE Healthcare). For immunoprecipitation, 500 μg total protein clear lysate was incubated with 20 μl of antibody (anti-6xHis, anti-Hsp90, anti-Cot, anti-Hsp90, antiHsp70, or anti-Grp78) or IgG chain C (Santa Cruz, as the negative control) for 2 h at 4°C, followed by incubating with protein G agarose (Invitrogen) (20 μl) for another 1 h at 4 °C. Conjugated agarose was harvested by centrifugation, washed (20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X100, 1 mM Na3VO4) twice and resuspended in 50 μl kinase buffer (20 mM MOPS pH 7.2, 50 mM β-glycerophosphate, 20 mM MgCl2, 10 mM NaF, 3 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM DTT). SDS-PAGE followed by Western blotting [anti-Cot(N17)] was used to confirm the quality of the immunoprecipitated protein. Kinase assays The verified immunoprecipitates (5 μl) were incubated with ATP (200 μM) and 1 μg MEK1 (Santa Cruz) in kinase buffer for 1 h at 30°C. Phosphorylated MEK1 was visualized by SDS-PAGE followed by Western blotting (probed with anti-phospho-MEK1/2 antibody). The membrane was stripped and reprobed with anti-MEK1/2 as the control. Fast activated cell-based ELISA (FACE) was performed following the manufacturer’s instructions (Active Motif) using either anti-MEK1/2 or anti-phospho-MEK1/2 as the primary antibody. The absorbance was recorded at 450 nm using a Benchmark microplate reader (Bio-Rad) (the OD595 was used to correct the measured readings for the cell number). 2D electrophoresis The 1D IEFs were performed on 18 cm IPG strips (GE Healthcare) rehydrated overnight with 375 μl solution containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 50 mM DTT, 3% (v/v) IPG buffer and 0.02% (w/v) bromophenol blue. The protein (ca. 50 μg per strip) was separated using a Binhui Wu & Rupert C. Wilmouth 45 Table 1. Isoelectric points (pI) and molecular weight (Mw) values of cellular proteins associated with Cot. Spot Approximate Approximate Empty Protein iden- SwissProt MOWS Cot130-399 Cot1-388 Cot1-413 Cot1-438 Cot1-457 Cot1-467 number Mw (kDa) pI value vector tification ID E score 1 2 3 4 200 120 150 85 5.75 6.05 6.35 5.15 5 69 5.15 6 58 5.05 + 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 52 44 62 65 63 63 45 46 49 34 44 37 31 26 24 5.35 5.15 5.85 5.95 5.95 6.05 5.80 6.00 6.75 6.35 7.35 5.45 5.85 5.25 5.20 + + + + + + + + + + + + + + + + Hsp90 P07900 101 Hsp70 P08107 277 Grp78 P11021 80 Tryptic peptides sequenced by MS/MS HFSVEGQLEFR GVVDSEDLPLNISR NPDDITNEEYGEFYK DAGVIAGLNVLR TTPSYVAFTDTER IINEPTAAAIAYGLDR NQVALNPQNTVFDAKR IEIESFYEGEDFSETLTR ITPSYVAFTPEGER + + + + + + + + + + + + + + + + + + + + + + + + + Ettan IPGphor II unit (GE Healthcare) with the following sequential parameters: 500 V for 500 Vhr; 1,000 V for 500 Vhr; 1,000−8,000 V gradient for 4,500 Vhr; 8,000 V for 64,000 Vhr. Prior to SDS-PAGE, the strips were soaked in 10 ml equilibration solution [6 M urea, 0.05 M TRIS pH 6.8, 30% (v/v) glycerol, 2% (w/v) SDS] supplemented with 10 mg ml−1 DTT for 15 min, followed by 20 ml equilibration solution supplemented with 25 mg ml−1 iodoacetamide and 0.02% (w/v) bromophenol blue for 15 min. The strips were laid on top of a 10% SDSPAGE gel and electrophoresis performed using a Protean II Xi unit (Bio-Rad) at 5°C at a constant current of 7 mA (per gel) for 1 h then 25 mA (per gel) for 6 h. The gel was silver-stained (GE Healthcare), scanned using ImageScanner II (GE Healthcare) and analyzed with ImageMaster software (GE Healthcare). Each gel was re-run at least two times and an identical pattern of mismatched spots (Table 1) was found in all repeats. Each gel slice was soaked in 20 μl of trypsin (3.33 ng μl−1, diluted by 50 mM ammonium bicarbonate) at 30°C for overnight or 37°C for 4 h. Peptides were extracted using 50% (v/v) acetonitrile with 0.5% (v/v) TFA (200 μl) followed by sonication (10 min) at 37°C. The solution was dried using a vacuum concentrator and redissolved in 50% (v/v) acetonitrile with 0.5% (v/v) TFA (1.5 μl). Peptides were desalted using a ZipTip (Millipore) and mixed with an equal volume of matrix solution [αcyano-4-hydroxycinnamic acid (5 mg) dissolved in 50 % (v/v) acetonitrile with 0.5% (v/v) TFA (1 ml)] and spotted (1 μl) onto a MALDI plate (384 opti-TOF, ABI). After air drying, spots were identified by a combination of MS followed by MS/MS (10 largest peaks) using an ABI 4800 MALDI-TOF/ TOF mass spectrometer. Peptides derived from trypsin were used as an internal standard. The MS/MS results were analyzed by searching the SwissProt protein database (restricted to Homo sapiens) using Mascot V1.9. Protein identification Gel slices encompassing the mismatched spots were cut-out and destained (15 min) in potassium ferricyanide (15 mM) and sodium thiosulphate (50 mM). The slices were then washed in Milli-Q water, soaked (5 min) in acetonitrile (85 μl) and dried using a vacuum concentrator (Eppendorf). Results and Discussion Expression of Cot kinase constructs A series of differ- 46 Proteomics Analysis of Proteins Associated with Cot A A B B C Fig. 1. A. HEK293 cells were transfected with empty vector or Cot constructs. Expression was determined by Western blots probed with anti-6xHis antibody. The same membrane was stripped and reprobed with anti-PDI as a control. B. Immunoprecipitation of the cell lysate (Cot1-467) with anti-6xHis, antiCot (N17), or anti-Cot (H212) was followed by Western blotting using anti-Cot (M20). C. Immunoprecipitation of the Cot contructs with anti-6xHis was followed by Western blotting using anti-Cot (M20). The same membrane was stripped and reprobed with anti-p50 (to detect p105). ent truncated constructs of Cot were created and transiently expressed in HEK293 cells: Cot130-399 (representing the kinase domain), Cot1-388 (the N-terminal and kinase domains), Cot1-413, Cot1-438 (containing the putative PEST sequence), Cot1-457 (containing both the PEST and degron sequences) and Cot1-467 (full-length). In order to determine the expression level of these constructs, the total cell lysate was probed with anti-6xHis 48 h posttransfection. Western blots [using anti-Cot (H212)] indicated that Cot1-413 was expressed at a higher level than the other constructs (Fig. 1A). However, it has been reported that the mRNA level, determined by RT-PCR, of Cterminally truncated Cot is similar to that of the fulllength protein (Gándara et al., 2003). In the same study, when the cells were treated with a proteasome inhibitor (MG132 or lactacystin), all the Cot constructs expressed protein at the same level. It is therefore possible that the rate of degradation of Cot1-413 may be less than the other constructs. Immunoprecipitation The pull-down efficiency (using Fig. 2. A. Kinase assay in vitro: phosphorylated MEK1 was visualized by Western blotting probed with anti-phosphoMEK1/2 antibody. The membrane was stripped and reprobed with anti-MEK1/2 as the control. The first lane (Mock) used kinase buffer in place of the immunoprecipitated protein as a negative control. B. Fast activated cell-based ELISA (FACE, kinase assay in vivo). Untreated, LPS-treated, TNF-α-treated cells were grown on a 96-well plate, fixed, quenched, and incubated with primary antibody (anti-MEK1/2 or anti-phosphoMEK1/2). The data were the average of three individual experiments and the error bars represent one standard deviation. expressed full-length Cot) of three antibodies was compared (Fig. 1B) and it was determined that anti-6xHis was optimal. The expressed Cot proteins were pulled down using this antibody and detected by Western blotting [using anti-Cot (M20)] (Fig. 1C). As p105 (NF-κB1) has been reported to be a physiological binding partner and an inhibitor of unactivated Cot, it was used as a control. The C-terminal half of p105 has been shown to bind to Tpl-2 at two sites: the C-terminal domain of Tpl-2 binds to the p105 ankyrin repeat region (497-534), and the kinase domain binds to the p105 death domain (808-892) (Beinke et al., 2003). The membrane was stripped and re-probed with anti-p50 (used to detected p105). Only Cot130-399 did not display a distinct p105 band which may indicate that binding to the kinase domain alone is not sufficient. Kinase assays To investigate the activity of the immunoprecipitated Cot proteins, in vitro kinase assays were performed. The proteins were incubated with the reported substrate MEK1 in presence of ATP. After Western blotting, they were probed with anti-phospho-MEK1/2 (Fig. 2A). All the Cot proteins appeared to be active, but Cot1-413 appeared to have slightly weaker activity than the other constructs despite its higher expression. An endotoxin-free plasmid maxi-prep kit (Qiagen) was used in an attempt to minimise the possibility of contamination of the E. coli- Binhui Wu & Rupert C. Wilmouth 47 2D gel electrophoresis The proteins that co-immunoprecipitated with Cot were separated by 2D gel electrophoresis in an attempt to identify potential binding partners in the absence of extracellular stimulation (Fig. 3). This experiment was repeated for each of the Cot constructs to allow the binding region to be determined. As the anti6xHis antibody is not completely specific to the expressed Cot proteins, HEK293 cells transfected with an empty vector were used as a control. Twenty-one protein spots were found in the Cot-transfected cell lysates that did not appear, or had substantially reduced intensity, in the control sample (Fig. 4 and Table 1). From MALDI-TOF/TOF analysis, three of these proteins were identified as heatshock protein Hsp70 and Hsp90 and glucose-regulated protein Grp78. The other spots have not yet been successfully identified, probably due to the quantity of protein being too low. From the pattern of association with the different Cot constructs (Table 1), it can also be determined that Hsp90 appears to bind to the Cot kinase domain. Fig. 3. 2D analysis of the proteins obtained from immunoprecipitation (anti-6xHis) of the cell lysate from HEK293 cells that had been transfected with the six different constructs and the empty vector as a control. Thirteen regions (A−M) have been highlighted which contain 21 mismatched spots. These gels represent examples from at least two repeats. The molecular weight positions were determined using markers (not shown), and the pI positions were determined from information supplied with the IPG gel strips (GE Healthcare). derived plasmids with LPS. In addition, in vivo kinase assays were performed using fast activated cell-based ELISA (FACE). The phospho-MEK1/2:total MEK1/ 2 ratio was measured for untreated, LPS- and TNF-α-treated transfected cells (Fig. 2B). All constructs demonstrated higher activity following LPS and TNF-α stimulation. This is in line with previous experiments (Caivano et al., 2003; Das et al., 2005) on wild-type Tpl-2, however our experiments additionally demonstrate that C-terminally truncated Cot behaves in a similar manner to the full-length protein. Interaction between Cot and Hsp90, Hsp70 and Grp78 The interaction between Cot and Hsp90, Hsp70 and Grp78 was further investigated using immunoprecipitation with both anti-Hsp90, anti-Hsp70 and anti-Grp78 (blotting with anti-Cot) and anti-6xHis (blotting with antiHsp90, anti-Hsp70 and anti-Grp78) (Fig. 5A). The pattern of association between the various Cot constructs and Hsp90, Hsp70 and Grp78 is identical to that determined from the 2D gel electrophoresis experiments (Table 1). Endogenous Cot was then immunoprecipitated from Hela cells (HEK293 cells express very low levels of endogenous Cot) and probed with Hsp90, p50 (to detect p105), Abin2 and Akt antibodies (Fig. 5B). This demonstrated that Hsp90 interacts with endogenous Cot together with the previously determined binding partners of Cot, Abin-2 (Lang et al., 2004) and Akt (Kane et al., 2002). The Western blot shows no interaction with p105 which may indicate that either the level of p105 is too low to be detected or that p105 has partially dissociated from Cot. Hsp90 has been shown to interact with around 100 proteins including many kinases involved in signalling pathways, including Akt (Sato et al., 2000), IKK (Broemer et al., 2004) and Raf (Stancato et al., 1993). Several Hsp90 inhibitors have been developed and are currently in clinical trials as anti-cancer therapeutic agents (Cullinan and Whitesell, 2006). Hsp70 is essential for the assembly of signalling protein:Hsp90 heterocomplexes, and this relates to the finding of this study that Hsp70 binds to Cot in the presence of Hsp90. The Hsp90/Hsp70 chaperone system is an important regulator of signal transduction pathways for steroid hormones receptors, kinase signalling, and transcription factors (Pratt and Toft, 2003). In agreement with the results from this study, Hsp90 has been demonstrated to bind directly to the kinase catalytic domain (Prince and Matts, 2004) and can directly affect 48 Proteomics Analysis of Proteins Associated with Cot Fig. 4. Two enlarged regions (C and D, corresponding to those shown in Fig. 3) from seven 2D gels (six different Cot constructs and the empty vector) showing the mismatched spots. A cluding Grp78 (Barati et al., 2006). It was demonstrated that the N-terminal domain of Cot/Tpl-2 and the kinase and/or C-terminal domains of Akt can physically interact and that this interaction is required for the Cot-mediated activation of NF-κB (Kane et al., 2002). It is therefore possible that Cot, Akt and the Hsp90/Hsp70/Grp78 chaperone proteins are part of a signalling complex along the NF-κB pathway. Acknowledgments We are grateful to Drs. Tang Kai and Newman Sze for their technical assistance in the mass spectrometry experiments. This work was supported by the Singapore Cancer Syndicate and Nanyang Technological University. B Fig. 5. A. HEK293 cells were transfected with empty vector or six Cot constructs. Immunoprecipitation of the cell lysate with anti-6xHis, anti-Hsp90, anti-Hsp70, or anti-Grp78 was followed by Western blotting using anti-Hsp90, anti-Hsp70, anti-Grp78 or anti-Cot (M20). B. Endogenous Cot was immunoprecipitated from Hela cell lysate using anti-Cot (H212). The pulled-down immunocomplex was probed with Hsp90, p50 (to detect p105), Abin-2 and Akt antibodies. Non-specific IgG was used as the negative control. kinase activity and modulate phosphorylation. Grp78 (also known as BiP) is a chaperone that is abundant in the endoplasmic reticulum and is a member of Hsp70 family (Mayer and Bukau, 1998). Akt appears to phosphorylate the chaperone proteins of the Hsp90/Hsp70 complex, in- References Aoki, M., Hamada, F., Sugimoto, T., Sumida, S., Akiyama, T., and Toyoshima, K. (1993). The human cot proto-oncogene encodes two protein serine/threonine kinases with different transforming activities by alternative initiation of translation. J. Biol. Chem. 268, 22723−22732. Barati, M.T., Rane, M.J., Klein, J.B., and McLeish, K.R. (2006). A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells. J. Proteome Res. 5, 1636−1646. Beinke, S., Deka, J., Lang, V., Belich, M.P., Walker, P.A., Howell, S., Smerdon, S.J., Gamblin, S.J., and Ley, S.C. (2003). NF-κB1 p105 negatively regulates TPL-2 MEK kinase activity. Mol. Cell. Biol. 23, 4739−4752. Broemer, M., Krappman, D., and Scheidereit, C. (2004). Requirement of Hsp90 activity for IκB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-κB activation. Oncogene 23, 5378−5386. Caivano, M., Rodriguez, C., Cohen, P., and Alemany, S. (2003). 15-Deoxy-Δ12,14-prostaglandin J2 regulates endogenous Cot MAPK kinase kinase 1 activity induced by lipopolysaccharide. J. Biol. Chem. 278, 52124−52130. Ceci, J.D., Patriotis, C.P., Tsatsanis, C., Makris, A.M., Kovatch, R., Swing, D.A., Jenkins, N.A., Tsichlis, P.N., and Copeland, N.G. (1997). Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. Genes Dev. 11, 688−700. Chiariello, M., Marinissen, M.J., and Gutkind, J.S. (2000). Multiple mitogen-activated protein kinase signaling pathways Binhui Wu & Rupert C. Wilmouth connect the Cot oncoprotein to the c-jun promoter and to cellular transformation. Mol. Cell. Biol. 20, 1747−1758. Cullinan, S.B. and Whitesell, L. (2006). Heat shock protein 90: a unique chemotherapeutic target. Semin. Oncol. 33, 457− 465. Das, S., Cho, J., Lambertz, I., Kelliher, M.A., Eliopoulos, A.G., Du, K., and Tsichlis, P. N. (2005). Tpl2/Cot signals activate ERK, JNK, and NF-κB in a cell-type and stimulus-specific manner. J. Biol. Chem. 280, 23748−23757. Dumitru, C.D., Ceci, J.D., Tsatsanis, C., Kontoyiannis, D., Stamatakis, K., Lin, J.H., Patriotis, C., Jenkins, N.A., Copeland, N.G., Kollias, G., et al. (2000). TNF-α induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell 103, 1071−1083. Erny, K.M., Peli, J., Lambert, J.F., Muller, V., and Diggelmann, H. (1996). Involvement of the Tpl-2/cot oncogene in MMTV tumorigenesis. Oncogene 13, 2015−2020. Gándara, M.L., López, P., Hernando, R., Castaño, J.G., and Alemany, S. (2003). The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity. Mol. Cell. Biol. 23, 7377−7390. Kane, L.P., Mollenauer, M.N., Xu, Z., Turck, C.W., and Weiss, A. (2002). Akt-dependent phosphorylation specifically regulates Cot induction of NF-κB-dependent transcription. Mol. Cell. Biol. 22, 5962−5974. Lang, V., Symons, A., Watton, S.J., Janzen, J., Soneji, Y., Beinke, S., Howell, S., and Ley, S.C. (2004). ABIN-2 forms a ternary complex with TPL-2 and NF-κB1 p105 and is essential for TPL-2 protein stability. Mol. Cell. Biol. 24, 5235−5248. Lin, X., Cunningham, E.T., Mu, Y., Geleziunas, R., and Greene, W.C. (1999). The proto-oncogene Cot kinase participates in CD3/CD28 induction of NF-κB acting through the NF-κBinducing kinase and IκB kinases. Immunity 10, 271−280. Makris, A., Patriotis, C., Bear, S.E., and Tsichlis, P.N. (1993). Genomic organization and expression of Tpl-2 in normal 49 cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion. J. Virol. 67, 4283−4289. Mayer, M.P. and Bukau, B. (1998). Hsp70 chaperone systems: diversity of cellular functions and mechanism of action. Biol. Chem. 379, 261−268. Pratt, W.B. and Toft, D.O. (2003). Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. 228, 111−133. Prince, T. and Matts, R.L. (2004). Definition of protein kinase sequence motifs that trigger high affinity binding of Hsp90 and Cdc37. J. Biol. Chem. 279, 39975−39981. Salmerón, A., Ahmad, T.B., Carlile, G.W., Pappin, D., Narsimhan, R.P., and Ley, S.C. (1996). Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase. EMBO J. 15, 817−826. Sato, S., Fujita, N., and Tsuruo, T. (2000). Modulation of Akt kinase activity by binding to Hsp90. Proc. Natl. Acad. Sci. USA 97, 10832−10837. Sourvinos, G., Tsatsanis, C., and Spandidos, D.A. (1999). Overexpression of the Tpl-2/Cot oncogene in human breast cancer. Oncogene 18, 4968−4973. Stancato, L.F., Chow, Y.H., Hutchison, K.A., Perdew, G.H., Jove, R., and Pratt, W.B. (1993). Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cellfree system. J. Biol. Chem. 268, 21711−21716. Tsatsanis, C., Patriotis, C., Bear, S.E., and Tsichlis, P.N. (1998). The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines. Proc. Natl. Acad. Sci. USA 95, 3827−3832. Vercauteren, F.G., Arckens, L., and Quirion, R. (2006). Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis. Amino Acids 33, 405−414.