Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK
* Author for correspondence (e-mail: michael.whitaker{at}ncl.ac.uk)
Accepted 19 August 2005
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Summary |
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Key words: MAP kinase, ERK1, Homodimerization, Mitotic cell cycle, Sea urchin embryos, Human cells
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Introduction |
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Protein dimerization has been found to be a common signalling motif during the transduction of extracellular signals. For MAP kinase pathways, it has been indirectly shown that phosphorylation of ERK2 facilitates the formation of dimers in vitro. By using equilibrium sedimentation, Khokhlatchev and colleagues have found that phosphorylated ERK2 sediments in vitro primarily as an 84 kDa species whereas nonphosphorylated ERK2 sediments as a mixture of a lesser amount of the 84 kDa and a greater amount of the 42 kDa species (Khokhlatchev et al., 1998). A model of dimerized kinase has been deduced from the crystal structure of phosphorylated ERK2, which reveals the physical basis for its dimerization (Canagarajah et al., 1997
). Phosphorylation causes conformational changes in the two flexible regions of the protein molecule, the activation loop and the C-terminal extension, and provides the surfaces for homodimerization. As a result, two ERK2 molecules bind via a hydrophobic zipper complemented by two ion pairs, one on each side of the zipper (for reviews, see Cobb and Goldsmith, 2000
; English et al., 1999
). It has been suggested that p38, ERK1 and JNK/SAPK also form homodimers (Khokhlatchev et al., 1998
). In addition, it has been shown that exogenous recombinant ERK2 dimerizes when transfected or microinjected into cells, that dimerization requires its phosphorylation and that dimerization is necessary for localization to the nucleus (Khokhlatchev et al., 1998
). However, despite the structural and cell biological evidence of the importance of dimerization, the in-vivo formation of dimers has not been demonstrated biochemically, nor have the relative kinase activities of phosphodimers and monomers been measured within cells. There has not until now been any evidence of dimer formation in whole-cell extracts, the usual method for determining ERK activity: the status and importance of dimerization in regulating kinase activity has not been investigated.
Here we use the ready availability of whole-cell extracts from sea urchin embryos to demonstrate that efficient ERK1 phosphodimer formation in vitro requires a soluble cofactor or cofactors. The phosphodimers are components of a high molecular weight protein complex in vivo and in vitro. We use in vitro activation/deactivation reactions to study the formation and disassembly of the ERK1 dimers. We also demonstrate that ERK1 forms homodimers in vivo by determining ways to isolate and detect them. Basal MAP kinase activity in vivo is maintained by homodimers formed by one phosphorylated and one unphosphorylated ERK1 molecule (a monophosphodimer). Peak MAP kinase activity is achieved by the accumulation of dimers formed of two phosphorylated ERK1 molecules (a bisphosphodimer). Free monomers are mainly unphosphorylated and inactive in vivo. We conclude that ERK1 bisphosphodimers are the overwhelmingly predominant form of highly active ERK1 in vivo, that basal ERK1 activity is generated by monophosphodimers and that phosphorylated monomers contribute very little to in vivo ERK1 activity.
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Materials and Methods |
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Expression vectors and purification of recombinant proteins
ERK1 expression vector encoding full length GST-tagged human ERK1 was a generous gift of B. Burgering (Utrecht, Holland). ERK1 dimerization-deficient mutant GST-PEHD-ERK1 was created by PEHD deletion situated five residues upstream of the activation loop. An identical ERK2 mutant that lacks the same four amino acids at the dimerization interface was unable to form dimers when microinjected (Khokhlatchev et al., 1998
). Two pRSET constructs encoding constitutively active (His)6-tagged hMEK R4F and (His)6-tagged kinase-inactive hMEK1 were kindly provided by J. Ferrell, Jr (Stanford, CA) (Sohaskey and Ferrell, 1999
) and originally constructed by N. Ahn (Mansour et al., 1996
; Mansour et al., 1994
). Bacterial strain Origami (DE3)pLysS (Novagen) was used for transformation with the various constructs described above and the cultures were grown at 20°C to an OD at 600 nm of 0.6 in 1 litre Terrific Broth, 125 µg/ml ampicillin and 25 µg/ml chloramphenicol. After induction of fusion protein synthesis by 0.1 mM IPTG, the bacterial cultures were grown for an additional 20 hours. GST-tagged hERK1 and
PEHD-ERK1 recombinant proteins were purified on GSTrap FF column following manufacturer protocol. Soluble (His)6-tagged MEK1 proteins were purified on a HiTrap Chelating HP column as previously described (Mansour et al., 1994
). All pure recombinant proteins were stored at 20°C in PBS with 10% glycerol for not more than one year. Longer storage results in aggregation, most likely due to their hydrophobic nature. Pure recombinant hCL100 phosphatase was a kind gift of S. Keyse (Dundee, UK).
Cell extracts, immunoprecipitation and western blotting
Cell extracts from sea urchin eggs and early embryos during mitotic cell cycle were prepared as described (Philipova and Whitaker, 1998). In short, eggs/embryos were homogenized on ice and, after repeated centrifugation at 15,700 g for 30 minutes, the soluble fractions were aliquoted, frozen and stored at 80°C. Extracts from HeLa cells grown in 10% FCS as controls or after a 10 minute induction with 100 ng/ml EGF were obtained by washing the cells three times with ice-cold sterile PBS and then lysing and collecting them in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 10 mM EDTA, 0.5 M NaCl, 10% glycerol, supplemented with phosphatase and protease inhibitors) on ice. High speed supernatants were used for immunoprecipitation and western blotting.
Immunoprecipitation was carried out as previously described (Philipova and Whitaker, 1998). Sea urchin or HeLa cell extracts containing 250 µg total cellular protein were used for immunoprecipitation with 1 µg anti-active ERK or anti-ERK antibody. For western blotting, the immune complexes or samples were not boiled (unless otherwise stated). Instead, they were left for 20 minutes at room temperature in Laemmli sample buffer and then frozen or used for electrophoresis and blotting. In some of the experiments carried out with recombinant human ERK1, the reducing agent was omitted from the sample buffer, as indicated in the text. When identical samples were probed with different antibodies, they were run in wide wells, then blotted and the blots were cut into strips. Each identical strip was incubated in different antibody; one strip was always used as a negative control.
In vitro protein kinase and protein phosphatase assays
In vitro MEK1 assays were carried out as previously described (Mansour et al., 1996); 0.5 nM of recombinant MEK1 R4F or kinase-inactive recombinant MEK1 (Mansour et al., 1996
; Mansour et al., 1994
; Sohaskey and Ferrell, 1999
) was used per reaction. Phosphatase assays were carried out in 50 mM Tris-HCl, pH 7.5, 2 µM Microcystin in the presence or absence of 2 mM Na3VO4 for 1.5 hours at 30°C and 0.007 nM of recombinant human CL100 (Lewis et al., 1995
) was used per reaction.
The experiments involving activation/inactivation of the recombinant GST-hERK1 or GST-PEHD-hERK1 protein were carried out as follows. For each sample 6 pM of purified recombinant protein was used. The whole amount of recombinant protein needed for one series of experiments was phosphorylated (see above) for 1 hour at 30°C and then bound to Glutathion-Sepharose (20 µl beads were used per sample) at room temperature for 30 minutes. The beads were washed twice in washing buffer WB (PBS, 0.1% Triton X-100, 2.5 mM MgCl2) supplemented with phosphatase inhibitors and then samples were aliquoted into separate tubes for different treatments. Incubation with cellular extract (15 µg or 75 µg total protein) was carried out for 15 minutes at room temperature and then beads were washed twice in WB. For phosphatase treatment, the sample was first washed in 50 mM Tris-HCl, pH 7.5, then dephosphorylated for 1.5 hours at 30°C and finally washed in WB. Another sample was washed in MEK1 kinase buffer, left overnight for additional MEK1 activation reaction at 4°C, then washed in WB. At each step a sample was taken and Laemmli buffer without ß-mercaptoethanol (ß-ME) was added at room temperature for electrophoresis. For kinase activity measurements, the same series of experiments were carried out, samples were washed in MAP kinase buffer (Philipova and Whitaker, 1998
) and protein kinase assays were carried out with MBP as substrate. Radioactivities of MBP were analysed by phosphor-imager. In the case of non-radioactive kinase assays (Fig. 1F), dephosphorylated MBP (Upstate Biotech) was used as substrate and phosphorylated MBP was detected by western blotting using phospho-specific MBP antibody (Upstate Biotech). Results shown are representative of three independent experiments.
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Results |
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We also took aliquots treated identically to those of Fig. 1A but, instead of blotting the samples, we washed them into MAP kinase buffer and measured the activities of the GST-hERK1 aliquots against a myelin basic protein (MBP) substrate. The results from one representative experiment are shown in Fig. 1B, beneath the relevant gel lane, together with the quantitated activities. Recombinant GST-hERK1 itself had very little activity (Fig. 1B, lane 1), which remained the same after incubation in sea urchin extract (Fig. 1B, lane 2). However, when activated with MEK alone for 1 hour, it showed around a tenfold activation over baseline (Fig. 1B, lane 3). Addition to the sea urchin embryo extract and incubation for 15 minutes at room temperature elevated the activity of the recombinant kinase a further tenfold (Fig. 1B, lane 4). A fivefold increase in the amount of the cell extract (to 75 µg total protein) did not increase the activity of the aliquot (not shown), indicating that soluble factors were not limiting and that basal cellular kinase activity was not contributing to the ERK1 activity we measured. Partial dephosphorylation of the protein using recombinant CL100 decreased its activity considerably; note that Fig. 1A, lane 5 indicates that the high molecular weight complex is resistant to dephosphorylation relative to the monomer. Interestingly, additional overnight activation at 4°C by MEK further doubled (Fig. 1B, lane 6) the activity of recombinant hERK1 in this aliquot relative to that activated for 1 hour at 30°C (Fig. 1B, lane 4). Thus, maximal phosphorylation and complex formation under these conditions leads to an MBP kinase activity about 20-fold higher than measured in the aliquot containing only phosphorylated monomeric GST-hERK1.
Two other sets of aliquots, identical to those run on lanes 2, 4, 5 and 6 or lanes 4, 5 and 6, were blotted and probed with anti-ERK (Fig. 1C) and anti-GST (Fig. 1D) antibodies, respectively. They confirmed that the high molecular mass active protein complexes are indeed ERK1 complexes and that the recombinant GST-hERK1 is one of their components. They also showed that treatment of the highly active aliquot with the dual-specificity CL100 ERK-phosphatase led to loss of GST-hERK1 from the high molecular weight complex and its appearance as a monomer (compare lanes 4 and 5 in Fig. 1C and D).
These results show that pure recombinant ERK1 can be activated in vitro without dimerization. However, a short incubation in cellular extract promotes the formation of ERK1 complexes with much higher activity. Dephosphorylation using a MAP kinase phosphatase specific for dual phosphorylated ERK reduces this activity, while additional overnight phosphorylation leads to the exclusive formation of highly active complexes with a further doubling of activity. Addition of reducing agent dissociates the high molecular weight complexes, but leaves the active dimers intact.
A dimerization deficient mutant, GST-PEHD-hERK1, in which four amino acids were deleted from the sequence of hERK1 (see Materials and Methods), was used in control experiments. A similarly-mutated ERK2 recombinant protein has been shown to be unable to dimerize (Khokhlatchev et al., 1998
). The mutant protein was successfully activated as a monomer, as shown by its order of magnitude increase (8-fold) in relative activity after activation by MEK in buffer (Fig. 1F, lanes 1 and 2), comparable to the 11-fold activation of the unmodified recombinant hERK1 (Fig. 1B, lanes 2 and 3). However, it did not crossreact with the anti-dual phosphorylated ERK antibodies, most likely due to an altered configuration as a result of the deletion of four amino acids in close proximity to the activation loop (Khokhlatchev et al., 1998
). Unlike the control GST-hERK1 protein (Fig. 1E,F, lanes 5), the mutant protein did not show any further increase in activity or dimerization (Fig. 1E,F, lanes 3) after incubation with low activity sea urchin cell extract. Thus ERK1 dimerization is crucial for the highly increased kinase activity of the protein complex formed by phosphorylated recombinant hERK1 in cellular extract.
ERK1 activity in vivo resides in two species of homodimer
ERK1 activity increases twice during the first mitotic cell cycle after fertilization in the sea urchin, once immediately after fertilization and again during mitosis (Philipova and Whitaker, 1998; Philipova et al., 2005a
; Philipova et al., 2005b
). The timing and extent of these changes is shown schematically for ease of orientation (Fig. 2A).
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Our previous measurements show that a basal level of ERK1 activity is always detectable throughout the cell cycle; the activity never falls to zero (Philipova and Whitaker, 1998) (Fig. 2A), consistent with reports elsewhere (Pouyssegur and Lenormand, 2003
). Our immunoprecipitation with the anti-active polyclonal antibody and subsequent blots with the anti-dual phosphorylated monoclonal antibody (Fig. 2B) demonstrate that the 91 kDa protein band contains an active ERK1 molecule phosphorylated on threonine and tyrosine even when MAP kinase activity is at basal levels, at first sight a paradox. Our postulate is that basal ERK1 activity in vivo is manifested by the 91 kDA homodimers as they are consistently detected throughout the cell cycle. These low activity homodimers (monophosphodimers) have the potential to become additionally phosphorylated/activated to form bisphosphodimers at times when peak activity is required for cell cycle progression.
Although our experiments with recombinant human ERK1 (Fig. 1) indicate that human ERK1 is capable of forming a complex with sea urchin proteins, it seemed entirely possible that dimerization to form active ERK1 might be a particular property of sea urchin ERK1. To test this postulate, we immunoprecipitated active ERK from HeLa cell extracts prepared from both normally growing cells and cells hyperstimulated with EGF for 10 minutes. We found a result identical to that in sea urchin: stimulation of ERK was associated with an increase in the protein band attributable to an ERK bisphosphodimer (Fig. 2C). Note that the shifted band above 91 kDa crossreacts with both anti-ERK and anti-active ERK antibodies on immunoblotting and is thus identified as ERK. Moreover, active ERK was again found in a high molecular weight protein complex under non-reducing condition and MEK again found to be a component of this complex (Fig. 2C, first and fourth panel). Treating samples at 95°C before electrophoresis led to loss of immunoreactivity to the anti-active ERK antibody, while immunoblotting of identical samples with an anti-ERK antibody demonstrated that the human phosphodimers were dissociated under these conditions, in marked contrast to ERK dimers in sea urchin embryos. These data demonstrate that active ERK is found predominantly as dimers in HeLa cells and that heat treatment of dimers leads to dissociation of the dimers and in addition caused a severe reduction in the immunoreactivity of ERK1 towards the anti-active ERK antibody.
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Immunodetection of the same samples with anti-active ERK monoclonal antibody (Fig. 3B) confirmed that the 91 kDa species and its shifted counterpart contained active ERK1 and that the monomeric form was inactive. We obtained very similar results with a different anti-active MAP kinase antibody (not shown). These reports support our postulate that the shifted band represents the additionally phosphorylated 91 kDa species. It is notable that treatment with MEK does not lead to the appearance of detectable active monomeric ERK1. Either the 91 kDa species is a preferential substrate for MEK or any active monomers rapidly dimerize to form additional 91 kDa complexes; both may occur.
To test whether the appearance of the shifted band would correspond to a higher MAP kinase activity of the complex, we washed identically treated samples in MAP kinase buffer and used them in kinase assays. As expected, the untreated and inactive kinase-treated immunoprecipitated ERK1 complexes showed relatively low activity, while activation by active MEK resulted in much higher kinase activity (Fig. 3C) that correlates with the appearance of the shifted bisphosphodimer band.
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The fractions from the final phenyl-Sepharose chromatography step were blotted independently with both the anti-ERK and anti-active ERK antibodies. Both dimers and monomers were detected in all active fractions using the anti-ERK antibody, whereas dimers were detected in all active fractions when probed with the anti-active ERK antibody (not shown). Fraction 23 of the column eluate was close to the peak of ERK1 activity and is shown as an example (Fig. 4A, a,b).
To demonstrate that the dimerized bands coeluted with the ERK1 monomer are indeed ERK1 dimers, we carried out in vitro dephosphorylation/phosphorylation of fraction 23. Treatment of the fraction with the recombinant dual-specificity CL100 ERK-phosphatase led to the loss of the shifted dimer band, as judged by western blotting as before (Fig. 4B,C). The loss was prevented by the inclusion of the phosphatase inhibitor vanadate in the incubation buffer (Fig. 4B,C). Treating the fraction with recombinant active MEK dramatically increased the intensity of the shifted band, whereas kinase-inactive MEK was ineffective (Fig. 4B,C). Comparison of the degree of labelling of the dimerized ERK1 in the kinase-inactive control with that of the same bands after treatment with active MEK implies recruitment of monomer to dimers; indeed, only a small amount of active monomer is detected under these conditions (Fig. 4C, MEK). Thus the previously purified ERK1 shows the same dimerization behaviour as the MAP kinase studied in cell cycle extracts.
In contrast to immunoprecipitated complexes, ERK1 dimers in whole cell extracts were very resistant to phosphatases (not shown), most probably owing to the activity of the upstream activator MEK in the high molecular weight active-ERK protein complex.
Newly-phosphorylated ERK1 accumulates in the active-active bisphosphodimers
As confirmation that the shifted band above 91 kDa represented phosphorylated ERK1, we tracked newly phosphorylated ERK1 during in vitro radioactive phosphorylation of purified ERK1. In interpreting these results, the reader should note the relative abundance of monomeric ERK: it is far in excess of the 91 kDa band and its shifted counterpart (Fig. 4Aa). We phosphorylated ERK1 using active MEK and took samples at 2, 4 and 6 minutes (Fig. 5). 32P accumulated only to a very small extent in the 44 kDa monomer. The 120 kDa band (corresponding to the active, shifted band) was most strongly labelled whereas the 91 kDa band showed labelling comparable or in slight excess to the monomer. Radioactivity is also associated with protein complexes and precipitates that do not enter the gel. Similar patterns were seen with both boiled (not shown) and unboiled samples, indicating that sea urchin homodimers do not disassemble when heated under reducing conditions, in contrast to those isolated from HeLa cells (Fig. 2C). Identical experiments using kinase-inactive MEK showed no accumulation of 32P in the proteins (not shown).
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The pattern of 32P incorporation clearly shows that newly phosphorylated protein accumulates as the 120 kDa bisphosphodimer. These data confirm that newly phosphorylated ERK1 does not remain monomeric, but is immediately incorporated into dimers as soon as it is phosphorylated and activated. They also imply that MEK may preferentially phosphorylate the 91 kDa protein rather then the monomer. Both events lead to accumulation of highly labelled bisphosphodimers.
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Discussion |
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Evidence for the existence of ERK1 in dimeric form
It is known that GST itself can dimerize (reviewed in Armstrong, 1997). However, the GST tag does not contribute to the dimerization of the recombinant ERK1 protein under our experimental conditions. The evidence can be summarized as follows: (1) Pure GST-hERK1 does not oligomerize if stored as indicated in Materials and Methods. It is stable as a 71 kDa protein; (2) it does not oligomerize after incubation with cell extract (Fig. 1A,C, lane 2) or after activation with MEK (Fig. 1A, lane 3); (3) it forms dimers with the sea urchin endogenous ERK1 that lack GST tags; (4) active ERK1, immunoprecipitated or purified from whole cell embryonic or HeLa extracts, is dimerized in the absence of GST-tags; (5) a GST-tagged dimerization-deficient mutant, GST-
PEHD-hERK1, does not dimerize and, moreover, does not show highly increased protein kinase activity after activation and incubation in cell extract, unlike the control GST-hERK1 protein. All these results exclude a role of the GST tag in dimer-formation and the additional 10- to 20-fold higher activity of the dimerized recombinant hERK1.
ERK1 monophosphodimers manifest basal ERK1 activity, whereas ERK1 bisphosphodimers are present when ERK1 is fully active
We took advantage of the fact that sea urchin embryos progress synchronously through the cell cycle and show two distinct episodes of ERK1 activation during the first cell cycle (Philipova and Whitaker, 1998) and compared the behaviour of active ERK1 during periods of basal and stimulated ERK1 activity. We infer that the 91 kDa active ERK1 monophosphodimer runs anomalously relative to its predicted 88 kDa molecular mass because of a gel shift due to phosphorylation. We found that a proportion of the 91 kDa band that represents the dimer was further shifted during stimulation and used this gel shift to identify the 91 kDa band as an active-inactive monophospho-ERK1 dimer and the shifted band as the active-active bisphospho-ERK1 dimer. Treatment with the MEK inhibitor U0126 in vivo prevents the formation of the bisphosphodimer and leads to abolition of ERK1 activation above the basal level (Philipova et al., 2005a
; Philipova et al., 2005b
). We conclude that basal ERK1 activity is due to the presence of the monophosphodimer and that further phosphorylation of this species gives rise to the 5-7-fold more active bisphosphodimer. We show that ERK in HeLa cell extracts exhibited identical behaviour.
ERK1 dimers represent a very small proportion of total cell ERK1
By comparing the relative amounts of ERK1 in its three forms (one monomeric and two dimeric) in whole cell extracts without immunoprecipitation, we determined that around 97% of ERK1 is present as the monomer, even when ERK1 activity is at its peak. Around 3% of cell ERK1 is found as the 91 kDa monophosphodimer, whereas the bisphosphodimer is rarely detectable in whole cell extracts, except by immunoprecipitation. By contrast, the amount of active ERK1 in each dimer was roughly comparable when ERK1 was most active in sea urchin embryos (Fig. 2B,C). Since each bisphosphodimer contains two dual-phosphorylated ERK1 monomers, this finding implies a ratio of monophosphodimers to bisphosphodimers of 2:1 (if we assume that each antibody binds to a single ERK1 molecule in the bisphosphodimer and that the antibodies have identical affinity for both types of dimers). Thus around one-third of the dimers are fully activated during mitosis, when the MBP kinase activity increases five- to sevenfold, suggesting that the bisphosphodimers have a 10-20-fold greater kinase activity than the monophosphodimers and in turn represent around 1% of total cell ERK1.
ERK1 dimerizes only in the presence of cellular protein(s), it forms a protein complex in vitro and also when isolated from whole-cell extracts
It has been reported on good grounds that dimerization of ERK2 does not affect its kinase activity: dimerization-defective mutants have a kinase activity comparable with wild-type. However, these observations were made on purified ERK2 in vitro; it has been shown to dimerize in simple buffers with a Kd of 7 nM (Khokhlatchev et al., 1998). We have not observed dimerization of activated ERK1 in simple buffers in vitro and therefore tested whether it would occur in the presence of other soluble cellular proteins. We have found that recombinant hERK1 will dimerize in a soluble sea urchin cell extract. At the moment, the only indication of the nature of the cofactor comes from preliminary experiments performed with two types of HeLa whole-cell extracts, obtained either after lysis or after homogenization. They indicate that the cofactor might be a membrane-associated protein, because extracts from homogenized cells are more efficient in promoting ERK1 homodimerization in vitro. An interesting and unanswered question is whether the cofactor contributes to the enhanced protein kinase activity. The experiments using a
PEHD mutant protein demonstrate that dimerization is critical for the highly increased activity of the protein complex formed by phosphorylated hERK1 in cellular extract. Further studies are required to understand the sequence of events leading to the formation of semi-active and fully active dimers, and to identify the cellular cofactor and its precise role in these events.
Our results show that in vivo the active homodimerized ERK1 is a member of a high molecular weight protein complex that also contains MEK. However, MEK is not associated with the ERK1 dimers that we were able to isolate under reducing conditions. It is known that ERK1 forms complexes in vivo (Tanoue and Nishida, 2002) and there are docking sites for MAP kinases on scaffold proteins, upstream kinases and MAP kinase phosphatases and also on substrates (Tanoue et al., 2000
), suggesting that elements of the MAP kinase signalling module may form a molecular machine (Morrison and Davis, 2003
) in which ERK dimers play a central role.
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Conclusion |
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Acknowledgments |
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References |
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