Involvement of the Activation Loop of ERK in the Detachment from Cytosolic Anchoring

Abstract

The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173–181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176–181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176–178 to alanines and is prevented by deletion of these residues or by substitution of residues 179–181 to alanines. On the other hand, residues 176–181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein. The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173–181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176–181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176–178 to alanines and is prevented by deletion of these residues or by substitution of residues 179–181 to alanines. On the other hand, residues 176–181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein. Involvement of the activation loop of ERK in the detachment from cytosolic anchoring.Journal of Biological ChemistryVol. 292Issue 21PreviewThis article has been withdrawn by the authors. One cell from the 12-h, -TPA panel was duplicated in the 6-h, +TPA panel in Fig. 1A. The 12-h, -TPA panel from Fig. 1A was duplicated in the ERK2, +TPA panel from Fig. 5. Also in Fig. 5, the cells shown in the ALC, -TPA panel were duplicated. The phosphorylated MBP panel from Fig. 4 was inappropriately manipulated. In Fig. 9, the ALC, -TPA and the 183A, +TPA panels were inappropriately manipulated. Because the original data are no longer available, in the interest of maintaining accuracy in the published scientific literature, the authors wish to withdraw this article. Full-Text PDF Open Access mitogen-activated protein kinase common docking domain cytosolic retention sequence extracellular signal-regulated kinase green fluorescence protein myelin basic protein MAPK/ERK kinase, MKP3, MAPK phosphatase-3 tetradecanoyl phorbol acetate epidermal growth factor polymerase chain reaction dithiothreitol Chinese hamster ovary polyacrylamide gel electrophoresis Among the key steps in the signaling mechanism of mitogen-activated protein kinase (MAPK)1 cascades are the changes in subcellular localization of their components upon extracellular stimulation (reviewed in Refs. 1Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3197) Google Scholar, 2Ferrell Jr., J.E. Trends Biochem. 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Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (165) Google Scholar), ERK1/2 (ERKs), and RSK1–3 (8Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar) translocate into the nucleus upon stimulation. Although it is still controversial whether the translocation of MEKs requires an active process (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (165) Google Scholar, 9Adachi M. Fukuda M. Nishida E. J. Cell Biol. 2000; 148: 849-856Crossref PubMed Scopus (173) Google Scholar), it is clear that MEKs are rapidly exported from the nucleus soon after their translocation. This rapid export from the nucleus is mediated by a CRM1-dependent nuclear export signal (10Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), which results in an apparent cytosolic localization of MEKs before and after stimulation (11Lenormand P. Brondello J.M. Brunet A. Pouyssegur J. J. Cell Biol. 1998; 142: 625-633Crossref PubMed Scopus (193) Google Scholar). ERKs and RSK1–3, on the other hand, are retained in the nucleus for longer times after stimulation. Interestingly, the proper subcellular localization of the various components has been shown to play an important role in regulating the physiological functions of the ERK cascade. Thus, prevention of nuclear localization of constitutively active MEK1/2 reduced its oncogenic potential (12Fukuda M. Gotoh I. Adachi M. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 32642-32648Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and nuclear localization of ERKs has been correlated with mitogenesis and neurite outgrowth in PC12 cells (13Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (805) Google Scholar). Moreover, prevention of the nuclear translocation of ERKs strongly inhibited gene transcription (14Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar), and RSK2 activity in the nucleus was found necessary for EGF-induced transcription of c-fos gene (15Swanson K.D. Taylor L.K. Haung L. Burlingame A.L. Landreth G.E. J. Biol. Chem. 1999; 274: 3385-3395Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In resting cells, both ERK1 and ERK2 are localized in the cytosol due to interaction with various anchoring proteins, including microtubules, phosphatases, and a MEK-dependent retention (14Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar, 16Reszka Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, M. Gotoh Y. Nishida E. EMBO J. 1997; PubMed Scopus Google Scholar, H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The with of these as (14Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar) or Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) is not upon However, most of the ERK to from their cytosolic upon stimulation and translocate into the This is rapid in P. C. Brunet A. Pouyssegur J. J. Cell Biol. PubMed Scopus Google Scholar), and of nuclear M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). One is a and the other is a active process that ERK J. M. M. E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The active translocation of ERKs to phosphorylation of of the on its (11Lenormand P. Brondello J.M. Brunet A. Pouyssegur J. J. Cell Biol. 1998; 142: 625-633Crossref PubMed Scopus (193) Google M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar, J. M. M. E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The translocated ERKs in the where a of ERKs can be after their activity has Y. Hanoch T. Y. D. Seger R. 2000; PubMed Scopus (43) Google Scholar, Y. Y. Rubinfeld H. D. Hanoch T. E. D. Seger R. 1997; PubMed Scopus Google Scholar). This that the nucleus is a major for of ERKs after translocation. has been that the ERK is to the cytosol by a mechanism that nuclear of MEKs M. Fukuda M. Nishida E. J. Cell Biol. 2000; 148: 849-856Crossref PubMed Scopus (173) Google Scholar). a sequence important for retention of ERKs in resting cells that we cytosolic retention sequence of ERK2 H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). This which is to play a role in interaction with Marshall C.J. 1994; PubMed Scopus Google Scholar, M. A. C. M. C. U. S. 1998; PubMed Scopus Google Scholar), residues in this were as a common docking domain for other as well T. Adachi M. T. Nishida E. Cell Biol. 2000; PubMed Scopus Google Scholar). However, the of functions to this and its of with other that other be in the of ERKs with Moreover, the residues and the mechanism responsible for the dissociation of ERKs from the anchoring are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in the of its subcellular localization. found that this dissociation is on residues 176–181 of ERK2, these residues do not play a significant role in the phosphorylation and activation of ERK2 upon stimulation. The dissociation was on the phosphorylation of ERK2 in its activation However, substitution of residues in this to alanines not the or shuttle of ERK2 through nuclear pores. These results that the translocation of ERK2 is through a dissociation from its cytosolic anchoring due to conformational in the activation The of ERK2 was into and downstream to the gene of a in which the of ERK2 is H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the were by H. A. Y. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), into the and fully to the of ERK2 were to alanines the a and a the of ERK2. 176–178 of ERK2 were to alanines the a and a the of ERK2. 176–178 of ERK2 were the and a of ERK2. 179–181 of ERK2 were to alanines the and a of ERK2. of ERK2 were to alanines the a and a the of ERK2. of ERK2 were with residues of MEK1 the a and a of The was of and of ERK2 were to alanines a with a and a with The was and of in this was to the a and a the of ERK2. MEK1 was as H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). was by with and The was and of The was by with the of and and reaction of and The various were into cells MEK1 was with the various of in the of was was with the various a of in the of was was as (7Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (165) Google Scholar). were with tetradecanoyl phorbol acetate and by fluorescence or by cells were for stimulation, the cells were with and with A. were with by and 4 The which cytosolic proteins, were and from were on by ERK was by with ERK or ERK protein was by MEK1 was with cells were and as The cell were to with MEK1 the were as (10Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) with and to with and the of ERK the were with with and with as H. A. Y. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The were to myelin basic protein phosphorylation as The of from the cells were with MBP and that in a of The phosphorylation reaction was to for after which it was by and for of MBP was by and In response to mitogenic stimulation, ERKs are translocated from the cytosol into the nucleus (8Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). this in subcellular localization we H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) green fluorescence protein to the of ERK2 that of in cells results in a nuclear of the protein both before and after stimulation. with MEK1 results in a cytosolic retention of the in resting cells, and this is mediated by residues of ERK2 This localization is rapidly upon mitogenic stimulation of the cells, which a dissociation of ERK2 from its cytosolic and its translocation into the the that govern the subcellular localization of ERK2, we the changes in the subcellular localization of after after the of the and after its these was the resting cells and stimulation a rapid nuclear translocation of the of In after after the of was and the of it was in the nucleus both before and after stimulation. These results can be by of a of by nuclear translocation of an of into the nucleus is responsible for the of protein in the However, stimulation is to the detachment of the ERKs from their cytosolic and a translocation that is after stimulation This to with anchoring as well as the of ERK2 translocation into the which were to be a and a M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). M. Gotoh Y. Nishida E. EMBO J. 1997; PubMed Scopus Google Scholar, H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), of and MEK1 in a cytosolic of the and this was by stimulation. the of ERK2, in which the and residues were by was with to nuclear translocation of the ERK2, that phosphorylation of ERK2 is essential for its nuclear translocation. we to the role of the activation loop of ERK2 in both its and nuclear translocation do we in the activation and as for ERK2 H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), the were to to their The substitution of to alanines substitution of 176–178 to alanines substitution of 179–181 to alanines substitution of to alanines were deletion of 176–178 to the role of these residues in the subcellular of the residues for of ERK2 and J. M. M. E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google and of the activation loop of ERK2 with the activation loop of an activation loop the activation and activity of the by into cells. after the cells were for and were with or the were to a as by their with ERK the of with that and were phosphorylated as as the ERK2, no phosphorylation was with 183A, and of the with not that it not be activated the However, with a phosphorylation on not on or and was with not These results that the is phosphorylated in response to on both and and the of by the was due to the of the The activity of the was by of the in cells with by an in kinase The activity with upon stimulation was to that of ERKs data not This activity was not or were and was with On the other hand, the activity of and was that of and and as well as not to the subcellular localization of the various activation in the of Thus, of the ERK2 was into cells with MEK1 and after was localized in the cytosol of resting cells and translocated into the nucleus after stimulation Fig. However, the that to was the and were to the cytosolic of the cells and M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). Interestingly, was localized in the nucleus both before and after stimulation was to the residues that to be important in the subcellular localization of ERK2 are residues which be phosphorylated and activated upon to translocate into the nucleus after this stimulation. the was the resting cells and rapidly translocated into the nucleus upon stimulation, that and the residues are important for the of ERK2 with MEK1 and are not essential for translocation. H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), of MEK1 for in nuclear of most to the of ERK2 were in the of resting cells both before and after not stimulation. These results that the to into the nucleus of resting cells, due to a mechanism M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). in the nucleus can be by the that to (11Lenormand P. Brondello J.M. Brunet A. Pouyssegur J. J. Cell Biol. 1998; 142: 625-633Crossref PubMed Scopus (193) Google Scholar), which can as a nuclear export for ERK2 M. Fukuda M. Nishida E. J. Cell Biol. 2000; 148: 849-856Crossref PubMed Scopus (173) Google Scholar), was not to nuclear export of the ERK2. whether the interaction of the ERK2 with anchoring is to their interaction with we the subcellular localization of the ERK2 after the of the MEK1 The results with this correlated with the were with MEK1 Thus, as as the and were the resting cells, that retention of of the On the other hand, and the which were not retained in the cytosol by were localized primarily in the nucleus in this that their to with retention the cells with and found that this nuclear translocation of and and localized in the in with the results with the MEK1 and of the cell and were to translocate into the nucleus in the of Thus, results that the retention to The detachment from these proteins, that from was on residues 176–181, and the substitution of residues 176–178 to alanines this The subcellular localization of the various ERK was after stimulation with and results to after stimulation not the that govern ERK dissociation from the cytosolic are and are on the of stimulation. the activation loop for their to with MEK1 in a Thus, and the various were into cells with for and or with for this the cells were and were with The of the were M. Gotoh Y. Nishida E. EMBO J. 1997; PubMed Scopus Google Scholar), and were to and with both and of was to of which was reduced upon with in the subcellular localization the that in a to the was On the other hand, 183A, and not seem to from MEK1 upon stimulation, as well as not with MEK1 to The the with MEK1 and the nuclear translocation of the ERK that the cytosolic interaction with MEK1 is the that the subcellular localization of the ERK2. been shown to as cytosolic for including Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and protein (14Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar). studied the of the various ERK2 to with the of MAPK (14Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google localization of was it was with in cells, and this localization was not by stimulation. to of residues of ERK2 to alanines H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google in the to the of ERK2 to residues T. Adachi M. T. Nishida E. Cell Biol. 2000; PubMed Scopus Google Scholar). However, the results with MEK1 with MKP3, the activation loop of ERK2 in a to the and localized to the cytosol both before and after stimulation These results the activation loop plays a role in the of ERK2 with it not to the of ERK2 to Moreover, in of the of ERK2 to from upon stimulation, these results that the activation loop is responsible for the dissociation of ERK2 from its cytosolic The of activation for the nuclear translocation of ERKs on the of residues in the activation loop of ERK2 for its dissociation from and translocation into the Here we a of that the activation loop of ERK2. in residues which are to the and a on the phosphorylation and activation of ERK2 and The that were were and which the and residues and as an control in studied the subcellular localization of the various activation loop of ERK2. has been M. Gotoh Y. Nishida E. EMBO J. 1997; PubMed Scopus Google Scholar) that MEKs with ERKs and in the However, of the of the the H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), it is that this is not mediated a protein J. C. J. 1998; PubMed Scopus Google Scholar). results as M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar), not from MEK1 and not translocate into the nucleus after stimulation the changes in residues 176–181 the subcellular localization of ERK2. Thus, residues 176–178 were to was no interaction with and this correlated with a nuclear of the ERK2 However, deletion of the residues in an with which a localization both before and after stimulation. These results are best by that these residues are important for the dissociation of ERK2 from Thus, as for the of with MEK1 is mediated by the residues of ERK2 H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the of to from MEK1 in response to stimulation can be by an of residues 176–178 to as a that dissociation from Substitution of the 176–178 residues to the activated of ERK2 and not the the was This is by the that substitution of these residues with residues a response as and not as not Moreover, with Fig. of the alanines the with is not is no of residues that dissociation and no dissociation of the that the of ERK2 was Interestingly, substitution of residues 179–181 to alanines an with MEK1 and it is that residues 176–181 are in the In to the involvement of residues 176–181 in the dissociation of ERK2 from these residues play a role in determining the of interaction the domain of ERKs to be to the in other it was T. Adachi M. T. Nishida E. Cell Biol. 2000; PubMed Scopus Google Scholar) that other be responsible for the of of the to the Thus, that residues 176–181 are important for the dissociation from MEK1 not from that these residues in the of of ERKs to The of the activation loop a of other of ERKs that were to be important for the ERK and These are residues in of ERKs A. Pouyssegur J. 1996; 272: PubMed Scopus Google Scholar), in the and of ERKs J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), of ERK2 Mol. Cell. Biol. PubMed Scopus Google Scholar), and residues H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), which residues and T. Adachi M. T. Nishida E. Cell Biol. 2000; PubMed Scopus Google Scholar) seem to play the most important role in the interaction with is clear that these residues with of MEK1 the are in of the ERK2 is that of ERK2 and MEK1 One of these is for the activation of ERK2 by MEK1 and the in the of the activation loop J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The other interaction the which not seem to play a significant role in the activation process of ERK2 H. Hanoch T. Seger R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Adachi M. T. Nishida E. Cell Biol. 2000; PubMed Scopus Google Scholar) in its subcellular localization and to Although is that ERK2 and MEK1 can with other M. Gotoh Y. Nishida E. EMBO J. 1997; PubMed Scopus Google Scholar, J. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), it is still that this interaction a protein as for ERK1 A. 1998; PubMed Scopus Google Scholar). In this the dissociation in not be from MEK1 from this protein. The mechanism of ERK2 translocation into the nucleus can be into In the the is from its cytosolic the of a shuttle through the nuclear The process of nuclear is through nuclear that are to with with shuttle through the nuclear by an active mechanism that requires a of Trends Biochem. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar). to M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar), we that the can translocate into the nucleus in a and Fig. and a and that the of the requires that both of translocation be mediated by an active in the nuclear in resting cells that the activation loop of ERK2 not play a role in its nuclear translocation. is that the responsible for the of translocation in a in resting cells and is activated by mitogenic stimulation of the cells. However, the translocation correlated with dissociation of the ERK2 from Although we a role of phosphorylation in the nuclear results that the translocation of ERK2 into the nucleus is primarily by its dissociation from MEK1 M. Fukuda M. Nishida E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). Thus, ERK2 from no signal is for the translocation to has been that of ERK2 is for its active nuclear and that phosphorylated ERK2 can with phosphorylated or ERK2 J. M. M. E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). on of phosphorylated ERK2, were the by the The of ERK2, was which the to upon stimulation, and into the cytosol of cells, not translocate into the it has been that these residues in and are for the translocation process of ERK2. However, in the in which these residues were to alanines in the of MEK1 and with MEK1 the was the cells, and stimulation its rapid nuclear translocation Thus, results that these residues or the in the in the translocation of ERK2 into the Although we the that these residues are important for the of ERK2, results that these residues do not in the nuclear translocation in it that in with other is important for the interaction of ERK2 with and its conformational rearrangement upon stimulation is essential for the dissociation of these In we in the activation loop of which to be important for the dissociation of ERK2 from of these residues upon phosphorylation to be in the mechanism of of ERK2 from its cytosolic and this can be mimicked by substitution of residues 176–178 to alanines. residues 173–181 not seem to play a significant role in the phosphorylation of ERK2 by that ERK2 into the nucleus in a in resting cells and a upon mitogenic stimulation. The residues of the activation loop of ERK2, including the do not play a role in these of results that the translocation of ERK2 into the nucleus is through the dissociation of ERK2 from cytosolic anchoring proteins, and this is mediated by residues in the activation loop of ERK2.