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ERK5 MAP kinase regulates neurogenin1 during cortical neurogenesis.

Cundiff P, Liu L, Wang Y, Zou J, Pan YW, Abel G, Duan X, Ming GL, Englund C, Hevner R, Xia Z - PLoS ONE (2009)

Bottom Line: We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1.Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1.Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

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S179 and S208 are required for Neurog1's transcriptional activity.A, Schematic representation of the various functional domains of Neurog1. Four putative proline-directed MAP kinase phosphorylation sites (PX1-2S/TP), S179, S201, S208 and T237 are present within the presumed transactivation domain in the C-terminus. B, Replacing S201 or T237 with alanine had no effect on Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. V: vector control. C, Replacing S179, S208, or both with alanine almost completely abolished Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. D, E, Western analysis demonstrating equal expression of wt Neurog1 and Neurog1 mutants in HEK293 cells. F, Replacing S179, S208, or both with alanine greatly attenuates Neurog1's ability to stimulate NeuroD2-luciferase activity in E16 cortical neuron cultures.
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pone-0005204-g003: S179 and S208 are required for Neurog1's transcriptional activity.A, Schematic representation of the various functional domains of Neurog1. Four putative proline-directed MAP kinase phosphorylation sites (PX1-2S/TP), S179, S201, S208 and T237 are present within the presumed transactivation domain in the C-terminus. B, Replacing S201 or T237 with alanine had no effect on Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. V: vector control. C, Replacing S179, S208, or both with alanine almost completely abolished Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. D, E, Western analysis demonstrating equal expression of wt Neurog1 and Neurog1 mutants in HEK293 cells. F, Replacing S179, S208, or both with alanine greatly attenuates Neurog1's ability to stimulate NeuroD2-luciferase activity in E16 cortical neuron cultures.

Mentions: Because ERK5 is a MAP kinase that can directly phosphorylate and regulate the activity of transcription factors [20], we postulated that ERK5 may regulate the transcriptional activity of Neurog1 through direct phosphorylation. A protein sequence analysis revealed two perfectly matched, putative proline-directed MAP kinase phosphorylation sites (PX 1-2 S/T P), S179 and S208, and two imperfect sites (S/T P), S201 and T237, within the C-terminus of Neurog1 (Fig. 3 A). Although mutations of S201 or T237 to non-phosphorylatable alanines had no effect on Neurog1's transcriptional activity (Fig. 3 B), replacing S179 or S208 with alanines almost completely abolished Neurog1's ability to initiate transcription in HEK293 cells (Fig. 3 C) and in cortical neurons (Fig. 3 F). The distinct effects of the four mutations on Neurog1's transcriptional activity were not due to differential expression of the mutant proteins (Fig. 3, D and E). These results suggest that Neurog1's transcriptional activity requires the function of S179 and S208. Furthermore, phosphorylation of S179 and S208 may regulate the transcriptional activity of Neurog1. Because the double mutant SA179/208 was as effective as, if not more potent than, the single mutants we focused our efforts on the double mutant for the remaining investigation.


ERK5 MAP kinase regulates neurogenin1 during cortical neurogenesis.

Cundiff P, Liu L, Wang Y, Zou J, Pan YW, Abel G, Duan X, Ming GL, Englund C, Hevner R, Xia Z - PLoS ONE (2009)

S179 and S208 are required for Neurog1's transcriptional activity.A, Schematic representation of the various functional domains of Neurog1. Four putative proline-directed MAP kinase phosphorylation sites (PX1-2S/TP), S179, S201, S208 and T237 are present within the presumed transactivation domain in the C-terminus. B, Replacing S201 or T237 with alanine had no effect on Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. V: vector control. C, Replacing S179, S208, or both with alanine almost completely abolished Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. D, E, Western analysis demonstrating equal expression of wt Neurog1 and Neurog1 mutants in HEK293 cells. F, Replacing S179, S208, or both with alanine greatly attenuates Neurog1's ability to stimulate NeuroD2-luciferase activity in E16 cortical neuron cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664926&req=5

pone-0005204-g003: S179 and S208 are required for Neurog1's transcriptional activity.A, Schematic representation of the various functional domains of Neurog1. Four putative proline-directed MAP kinase phosphorylation sites (PX1-2S/TP), S179, S201, S208 and T237 are present within the presumed transactivation domain in the C-terminus. B, Replacing S201 or T237 with alanine had no effect on Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. V: vector control. C, Replacing S179, S208, or both with alanine almost completely abolished Neurog1's ability to stimulate NeuroD2-luciferase in HEK293 cells. D, E, Western analysis demonstrating equal expression of wt Neurog1 and Neurog1 mutants in HEK293 cells. F, Replacing S179, S208, or both with alanine greatly attenuates Neurog1's ability to stimulate NeuroD2-luciferase activity in E16 cortical neuron cultures.
Mentions: Because ERK5 is a MAP kinase that can directly phosphorylate and regulate the activity of transcription factors [20], we postulated that ERK5 may regulate the transcriptional activity of Neurog1 through direct phosphorylation. A protein sequence analysis revealed two perfectly matched, putative proline-directed MAP kinase phosphorylation sites (PX 1-2 S/T P), S179 and S208, and two imperfect sites (S/T P), S201 and T237, within the C-terminus of Neurog1 (Fig. 3 A). Although mutations of S201 or T237 to non-phosphorylatable alanines had no effect on Neurog1's transcriptional activity (Fig. 3 B), replacing S179 or S208 with alanines almost completely abolished Neurog1's ability to initiate transcription in HEK293 cells (Fig. 3 C) and in cortical neurons (Fig. 3 F). The distinct effects of the four mutations on Neurog1's transcriptional activity were not due to differential expression of the mutant proteins (Fig. 3, D and E). These results suggest that Neurog1's transcriptional activity requires the function of S179 and S208. Furthermore, phosphorylation of S179 and S208 may regulate the transcriptional activity of Neurog1. Because the double mutant SA179/208 was as effective as, if not more potent than, the single mutants we focused our efforts on the double mutant for the remaining investigation.

Bottom Line: We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1.Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1.Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

Show MeSH
Related in: MedlinePlus