Differential regulation of mitogen-activated protein kinases ERK1/2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons

Abstract

Activation of the extracellular signal-regulated kinase 1 (ERK1) and ERK2 by neurotrophins, neuronal activity, or cAMP has been strongly implicated in differentiation, survival, and adaptive responses of neurons during development and in the adult brain. Recently, a new member of the mitogen-activated protein (MAP) kinase family, ERK5, was discovered. Like ERK1 and ERK2, ERK5 is expressed in neurons, and ERK5 stimulation by epidermal growth factor is blocked by the MAP kinase/ERK kinase 1 (MEK1) inhibitors PD98059 and U0126. This suggests the interesting possibility that some of the functions attributed to ERK1/2 may be mediated by ERK5. However, the regulatory properties of ERK5 in primary cultured neurons have not been reported. Here we examined the regulation of ERK5 signaling in primary cultured cortical neurons. Our data demonstrate that, similar to ERK1/2, ERK5 is activated by neurotrophins including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4. BDNF stimulation of ERK5 required the activity of MEK5. Surprisingly, ERK5 was not stimulated by cAMP or neuronal activity induced by glutamate or membrane depolarization. In contrast to ERK1/2, ERK5 strongly activated the transcriptional activity of myocyte enhancer factor 2C (MEF2C) in pheochromocytoma 12 (PC12) cells and was required for neurotrophin stimulation of MEF2C transcription in both PC12 cells and cortical neurons. Furthermore, ERK1/2, but not ERK5, induced transcription from Elk1 and the cAMP/ Ca(2+) response element in PC12 cells. Our data suggest that mechanisms for regulation of ERK5 and downstream transcriptional pathways regulated by ERK5 are distinct from those of ERK1/2 in neurons. Furthermore, ERK5 is the first MAP kinase identified whose activity is stimulated by neurotrophins but not by neuronal activity.

Fig. 1.

Neurotrophins induce ERK5 and ERK1/2 phosphorylation in cortical neurons. A, Dose–response relationship of BDNF stimulation of ERK5 and ERK1/2 phosphorylation is shown. At DIV5, cortical neurons were treated with 0, 2, 5, 10, 25, or 50 ng/ml BDNF for 1 hr. Cell lysates from human embryonic kidney 293 cells transiently transfected with a constitutively active MEK5 and a wild-type ERK5 were used as a positive control (+) for the ERK5 phosphorylation shift. B, Kinetics of BDNF stimulation of ERK5 and ERK1/2 phosphorylation is shown. At DIV5, cortical neurons were treated with 10 ng/ml BDNF for the indicated times.C, NT-3 and NT-4 also induce ERK5 and ERK1/2 phosphorylation. At DIV5, cortical neurons were treated with 10 ng/ml NT-3 or NT-4 for 0.5, 2, or 12 hr. Cell lysates were prepared, and 20 μg of total protein was submitted to Western analysis using antibodies recognizing ERK5 (Abe et al., 1996) or phosphorylated (p) ERK1/2. Phosphorylation of ERK5 was observed as a shift in ERK5 mobility, indicative of ERK5 activation. The anti-phospho-ERK1/2 antibody recognizes the phosphorylated and activated ERK1/2, indicative of ERK1/2 activation. Data are representative of four (A, B) or three (C) independent experiments. Thepositions of molecular weight markers are indicated on the right of the figures.

Fig. 2.

BDNF stimulation of ERK5 phosphorylation requires receptor tyrosine kinase activity. Cortical neurons (DIV5–DIV6) were pretreated with 0, 2, or 5 μm K252a for 30 min and then stimulated with 10 ng/ml BDNF for 0.5, 1, or 2 hr as indicated. Phosphorylation (p) of ERK5 and ERK1/2 was measured by Western analysis as described in Figure 1. Similar results were obtained in two independent experiments.

Fig. 3.

BDNF activates ERK5 in cortical neurons. Cortical neurons (DIV5–DIV6) were treated with 10 ng/ml BDNF for various times. Three hundred micrograms of total protein were used to measure ERK5 activity by the autophosphorylation assay. A, A representative autoradiograph of the ERK5 autophosphorylation kinase assay. B, Quantitation of ERK5 autophosphorylation.Inset, A more detailed profile of ERK5 activation at early time points. Data are the average of five to seven experiments. Error bars represent SEM.

Fig. 4.

MEK5 is activated by BDNF and is required for BDNF stimulation of ERK5 in cortical neurons. A, Endogenous MEK5 is activated by BDNF. At DIV5, cortical neurons were treated with 10 ng/ml BDNF for various times. Three hundred micrograms of total protein were used for an MEK5 immune complex kinase assay with truncated GST–ERK5(M) as the substrate. Inset, A more detailed profile of MEK5 activation at early time points is shown. Data shown are averages of two independent experiments. Error bars represent SEM. B, C, Expression of a dominant-negative MEK5 blocks BDNF stimulation of ERK5 (B) but not ERK2 (C). Cortical neurons (DIV3; 2 × 10⁶ cells/35 mm dish) were cotransfected with 2 μg each of plasmid DNA encoding a wild-type Flag-tagged ERK5 (ERK5wt) or ERK2 (ERK2wt), a dominant-negative HA-tagged MEK5 (MEK5DN), or a vector control (pCMV5) as indicated. Two days later, cells were treated with 10 ng/ml BDNF for 1 hr. Three hundred micrograms of total protein were used for immunoprecipitation with anti-Flag antibody. The transfected ERK5 and ERK2 kinase activities in the precipitates were assayed using GST–MEF2C or MBP as the respective substrates. Data shown are averages of three independent experiments. Error bars represent SEM.

Fig. 5.

cAMP, KCl, and glutamate activate ERK1/2 but not ERK5 in cortical neurons. Cortical neurons (DIV5) were treated with vehicle control, 55 mm KCl (A), 100 μm glutamate (B), or 2, 5, 10, or 50 μm forskolin (C) that increases intracellular cAMP for the indicated times. For a positive control, neurons were treated with BDNF (10 ng/ml; 1 hr). Phosphorylation (p) of ERK5 and ERK1/2 was measured by Western analysis as described in Figure 1. Similar results were obtained in three independent experiments. Fsk, Forskolin.

Fig. 6.

ERK5 autophosphorylation is not increased by KCl, glutamate, or forskolin treatment of cortical neurons. Cortical neurons (DIV5–DIV6) were treated with 10 ng/ml BDNF, 55 mm KCl, 30 μm glutamate, or 2 μm forskolin for various times. Three hundred micrograms of total protein were used to measure ERK5 activity by the autophosphorylation assay. Similar results were obtained with 50 μm forskolin. Data shown are the averages of four to seven experiments. Error bars represent SEM.

Fig. 7.

MEF2C-transactivating activity is stimulated by NGF and ERK5 in PC12 cells. PC12 cells were transfected with a Gal4–luciferase reporter gene (0.2 μg/3 wells) and an expression vector for Gal4–MEF2C fusion protein (0.4 μg/3 wells) to measure MEF2C transcriptional activity. An EF promoter-driven LacZ expression vector was cotransfected in all cases to normalize for transfection efficiency. A, MEF2C-mediated transactivation is preferentially stimulated by constitutive activation of the ERK5 pathway. To activate ERK5 or ERK1/2, cells were cotransfected with expression vectors (0.1 μg each/3 wells) encoding a constitutively active MEK5 (MEK5CA) with a wild-type ERK5 (ERK5wt) or a MEK1CA with anERK2wt, respectively. Data shown are the averages of 12 independent experiments ± SEM. B, MEF2C-mediated transcription is activated by NGF via an ERK5-dependent mechanism. To block ERK5 signaling, PC12 cells were transiently transfected with a dominant-negative MEK5 (0.9 μg/4 wells; MEK5DN) together with a dominant-negative ERK5 (0.9 μg/4 wells;ERK5DN) or the corresponding vector controls. Cells were treated with 50 ng/ml NGF (+NGF) or vehicle control (−NGF) for 6 hr. Data shown are the averages of three independent experiments ± SEM.Luc, Luciferase.

Fig. 8.

BDNF activates MEF2C in cortical neurons that require ERK5 signaling. A, MEF2C-mediated transcription is activated by BDNF via an ERK5-dependent mechanism. Cortical neurons (DIV4–DIV5; 0.5 × 10⁶ cells/well) were transiently transfected with a Gal4–luciferase reporter gene (1.4 μg/4 wells) and an expression vector for Gal4–MEF2C fusion protein (0.9 μg/4 wells) to measure MEF2C transcriptional activity. To block ERK5 or ERK1/2 signaling, neurons were cotransfected with a dominant-negative MEK5 (0.9 μg/4 wells; MEK5DN) together with a dominant-negative ERK5 (0.9 μg/4 wells;ERK5DN) or with a dominant-negative MEK1 (1.8 μg/4 wells; MEK1DN), respectively. The corresponding vectors were used as controls. Cells were treated with 10 ng/ml BDNF (+BDNF) or vehicle control (−BDNF) for 6 hr. Data are representative of quadruplicate determinations from three independent experiments.B, KCl-activated CRE transcription is inhibited by the dominant-negative MEK1 used in A. To confirm that theMEK1DN used in A functioned properly as a dominant negative, cortical neurons were cotransfected with a CRE–luciferase reporter (2.4 μg/4 wells), a MEK1DN, or its vector control (1.8 μg/4 wells). Cells were treated with 55 mm KCl for 6 hr. Data shown are the averages of quadruplicate determinations. For both A andB, an EF promoter-driven LacZ expression vector (0.55 μg/4 wells) was cotransfected to normalize for transfection efficiency, and error bars indicate SEM. Luc, Luciferase.

Fig. 9.

ERK5 does not stimulate Gal4–Elk1 transactivation or CRE-mediated transcription in PC12 cells. A, Transactivation of Elk1 is increased after NGF treatment or stimulation of the ERK1/2 but not the ERK5 pathway. PC12 cells were transfected with a Gal4–luciferase reporter gene (0.2 μg/3 wells) and an expression vector for the Gal4–Elk1 fusion protein (0.4 μg/3 wells). To activate ERK5 or ERK1/2, cells were cotransfected with expression vectors encoding a constitutively active MEK5 (0.2 μg/3 wells;MEK5CA) with a wild-type ERK5 (0.6 μg/3 wells;ERK5wt) or a MEK1CA (0.6 μg/3 wells), respectively. B, NGF treatment or constitutive activation of ERK1/2, but not ERK5, stimulates CRE-mediated transcription. PC12 cells were transfected with a CRE–luciferase reporter (1.2 μg/3 wells) to measure transcription initiated from CRE. To activate ERK5 or ERK1/2, cells were cotransfected with expression vectors encoding MEK5CA (0.1 μg/3 wells) and ERK5wt (0.3 μg/3 wells) or MEK1CA(0.1 μg/3 wells) and ERK2wt (0.3 μg/3 wells), respectively. An EF promoter-driven LacZ expression vector was cotransfected in all cases to normalize for transfection efficiency. When indicated, cells were treated with NGF (50 ng/ml) for 6 hr. Data shown are the averages of triplicate determinations ± SEM. Similar results were obtained in three to four independent experiments.Luc, Luciferase.