Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation - PubMed (original) (raw)

Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation

J Xing et al. Mol Cell Biol. 1998 Apr.

Abstract

The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

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Figures

FIG. 1

FIG. 1

All three RSK family members are activated by NGF. PC12 cells were either untreated or treated with NGF (30 ng/ml) for 10 min. RSK1, RSK2, and RSK3 were immunoprecipitated from extracts of untreated or NGF-treated PC12 cells with antibodies specific for RSK1, RSK2, or RSK3 respectively. RSK kinase activities in the immune complexes were determined by an in vitro kinase assay with CREBtide as a substrate. The fold activation is the ratio of kinase activity from NGF-treated cells to that from untreated cells. Data are from three separate experiments. Mean values are plotted; error bars represent standard errors of the mean (SEM).

FIG. 2

FIG. 2

All three RSK family members phosphorylate CREB Ser-133 in response to growth factor stimulation. (A) COS cells were transfected with expression constructs for either HA-RSK1, HA-RSK2, or HA-RSK3. Cells were either left untreated or treated with EGF for 10 min. HA-tagged RSKs were immunoprecipitated from lysates of untreated or EGF-treated transfected COS cells with a monoclonal anti-HA antibody (12CA5). Kinase activities were determined by an in vitro kinase assay with CREBtide as a substrate. Fold activation indicates the ratio of kinase activity from EGF-treated cells to that from untreated cells. Data are from three separate experiments. Error bars represent SEM. (B) COS cells were transfected with 1 μg of CMV-Gal4-CREB together with 10 μg of pMT vector (lanes 1 and 2), pMT2-HA-RSK1 (lanes 3 and 4), pMT2-HA-RSK2 (lanes 5 and 6), or pMT-HA-RSK3 (lanes 7 and 8). Transfected COS cells were left untreated (lanes 1, 3, 5, and 7) or treated for 10 min with EGF (30 ng/ml) (lanes 2, 4, 6, and 8). Lysates of transfected COS cells were separated by SDS-PAGE and immunoblotted with anti-PCREB (upper panel) or anti-Gal4 (lower panel) antibodies.

FIG. 3

FIG. 3

Inhibition of the MEK/ERK/RSK pathway leads to decreased NGF-stimulated CREB Ser-133 phosphorylation. (A) PC12 cells were either not pretreated, preincubated for 1 h with PD 098059 (100 μM), or preincubated for 1 h with SB 203580 (5 μM). The cells were then either lysed directly or treated with NGF (30 ng/ml) for 10 min before lysis. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-ERK (top) or anti-RSK2 antibodies (middle). Cells were either left untreated, or treated for 10 min with NGF (30 ng/ml). RSK2 was immunoprecipitated from cell lysates, and kinase activity was determined by an in vitro kinase assay with CREBtide as the substrate (bottom). Data are means from three independent experiments. Error bars represent SEM. (B) PC12 cells were either not pretreated or pretreated for 1 h with PD 098059 (100 μM). The cells were then either untreated or treated with NGF (30 ng/ml) for the indicated periods. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-PCREB antibodies.

FIG. 4

FIG. 4

Growth factor-induced pp70S6K activation is not sufficient to stimulate CREB Ser-133 phosphorylation. HepG2 cell lines stably expressing wild-type and mutant versions of PDGFR were serum starved for 2 days and either left untreated (lanes 1, 3, 5, 7, and 9) or treated for 10 min with PDGF (20 ng/ml) (lanes 2, 4, 6, 8, and 10). Cell lysates were separated by SDS-PAGE and immunoblotted with anti-PCREB (top) or anti-CREB (bottom) antibodies. The HepG2 lines were stably transfected with the empty pLXSN vector (N), the wild-type PDGFR (wt PDGFR), a mutant PDGFR in which Y740, Y751, Y771, Y1009, and Y1021 were replaced by phenylalanine (F5), the F5 mutant in which F740 and F751 were changed back to tyrosines (Y740/Y751), and a mutant PDGFR in which Y740 and Y751 were changed to phenylalanine (F740/F751).

FIG. 5

FIG. 5

p38 MAPK is activated in response to growth factor stimulation. (A) (Left) Lysates of PC12 cells that either were untreated or had been treated for 10 min with NGF (30 ng/ml) or for 30 min with anisomycin (20 μg/ml) were separated by SDS-PAGE and immunoblotted with anti-phospho-p38 MAPK (top) or anti-phospho-ERK antibodies (bottom). (Right) Lysates of NIH 3T3 cells that were untreated or treated for 10 min with EGF (30 ng/ml), or for 15 min with sorbitol (0.3 M) were separated by SDS-PAGE and immunoblotted with anti-phospho-p38 MAPK (top) or anti-phospho-ERK (bottom) antibodies. (B) p38 MAPK was immunoprecipitated from lysates of PC12 cells that were untreated (lane 1), treated with UV irradiation (lane 2), or treated with NGF (30 ng/ml) for various periods (lanes 3 to 8). p38 MAPK activity in the immune complex was measured by an in vitro kinase assay with GST-ATF2 protein as a substrate. The reaction products were examined by SDS-PAGE and autoradiography.

FIG. 6

FIG. 6

NGF does not stimulate JNK activity. (A) PC12 cells were untreated, treated with NGF (30 ng/ml) for various periods as indicated, or treated with UV irradiation. Cell lysates were separated by SDS-PAGE and immunoblotted with P-JNK antibodies. (B) JNK was immunoprecipitated from lysates of PC12 cells that were untreated, treated with NGF for various periods as indicated, or treated with UV irradiation. JNK activity was measured by an in vitro kinase assay with GST-c-Jun as a substrate. The reaction products were separated by SDS-PAGE and subjected to autoradiography (top). Kinase activities were quantified with a PhosphorImager in arbitrary units (bottom).

FIG. 7

FIG. 7

Activation of the p38 MAP kinase pathway leads to CREB Ser-133 phosphorylation in vivo. (A) COS cells were transfected with 1 μg of CMV-Gal4-CREB, together with either the empty vectors (pCMV6 and pcDNA3) or pCMV6-p38 and pcDNA3-MKK6(Glu) (constitutively active) or pcDNA3-MKK6(KA) (dominant negative). Two days after transfection, cell lysates were prepared, separated by SDS-PAGE, and immunoblotted with anti-PCREB (top) or anti-Gal4 (bottom) antibodies. (B) PC12 cells were either not pretreated or pretreated for 1 h with SB 203580 (5 μM) and were then either left untreated or treated for 10 min with NGF (30 ng/ml). Cell lysates were separated by SDS-PAGE and immunoblotted with an anti-PCREB antibody.

FIG. 8

FIG. 8

MAPKAP kinase 2 is activated by NGF. PC12 cells were not pretreated, pretreated for 1 h with PD 098059 (100 μM), or pretreated for 1 h with SB 203580 (5 μM). The cells were then left untreated, treated for 10 min with NGF (30 ng/ml), or treated with UV irradiation. MAPKAP kinase 2 was immunoprecipitated from cell lysates, and its kinase activity was determined by an in vitro kinase assay with CREBtide as a substrate. Data are means from three independent experiments. Error bars represent SEM.

FIG. 9

FIG. 9

NGF-induced CREB Ser-133 phosphorylation is mediated by the ERK/RSK and p38/MAPKAP kinase 2 pathways. PC12 cells were not pretreated, preincubated for 1 h with PD 098059 (100 μM), or preincubated for 1 h with SB 203580 (5 μM). The cells were then lysed directly, treated with NGF (30 ng/ml), or treated with UV irradiation before lysis. Cell lysates were separated by SDS-PAGE and immunoblotted with an anti-PCREB antibody.

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