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First published online 14 March 2006
doi: 10.1242/jcs.02837

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Protein kinase C-mediated proteasomal degradation of MAP kinase phosphatase-1 contributes to glutamate-induced neuronal cell death

System-Biodynamics NCRC, National Research Laboratory of Molecular Neurophysiology and Division of Molecular and Life Science, Pohang University of Science and Technology, Hyoja dong, San31, Pohang, 790-784, South Korea

View larger version (49K): [in a new window]   Fig. 1. ERK1/2 contributes to glutamate-induced cell death in HT22 cells. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. Cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies specific for phospho-ERK1/2 (pERK), phospho-JNK (pJNK), or phospho-p38 (pp38) MAPK. Membranes were stripped and reprobed for total ERK1/2, JNK or p38 as a control. Shown are blots from a representative experiment out of four separate experiments. (B) HT22 cells were incubated with 5 mM glutamate for 12 hours in the presence or absence of 20 µM U0126 (Glu/U0126), 10 µM SB202190 (Glu/SB) or 20 µM SP600125 (Glu/SP). Shown are representative light microscopic images. Results from the MTT assay are also presented. Each bar is a mean ± s.d. value from four separate experiments. (C) HT22 cells were exposed to 5 mM glutamate for 7 hours in the presence or absence of 20 µM U0126. Phosphorylation of ERK1/2 in whole cell extracts was determined by immunoblotting with an antibody against phospho-ERK1/2. Blots shown are representative of four separate experiments. (D) Representative photomicrographs of apoptotic cells treated with or without 5 mM glutamate for 12 hours in the presence or absence of 20 µM U0126. (E) HT22 cells were incubated with 5 mM glutamate for the indicated times. Cell extracts were immunoblotted with an antibody specific for phospho-ERK1/2 or phospho-MEK1/2. Membranes were stripped and reprobed for actin as a control. Results were reproducible, and blots shown are representative of three separate experiments. (F) HT22 cells were exposed to 5 mM glutamate, and then 20 µM U0126 was added either simultaneously or at the indicated times after the addition of glutamate. Cell viability was analyzed after 12 hours by the MTT conversion assay. Results are mean ± s.d. value from four separate experiments.

View larger version (26K): [in a new window]   Fig. 2. Glutamate induces activation and tyrosine phosphorylation of PKC in HT22 cells. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. Cell extracts were subjected to immunoprecipitation with anti-PKC. An in vitro immune complex kinase assay were performed using histone H1 as a substrate. The immunoprecipitates were also analyzed by immunoblotting with anti-PKC. (B) HT22 cells were incubated with 5 mM glutamate for the indicated times. Cells were then harvested, and immunoprecipitation of PKC was performed with an anti-PKC antibody as described in the Materials and Methods. Membranes were immunoblotted with anti-phosphotyrosine antibody (pTyr) and/or anti-PKC antibodies. The relative kinase activities and levels of tyrosine phosphorylation of PKC were quantified by densitometric analyses, and normalized to the level of total PKC (lower panel in A and B). Data represent an average fold increase as compared with controls (mean ± s.d.). *P<0.05 and **P<0.01, values compared with zero time. (C) HT22 cells were incubated with 5 mM glutamate for the indicated times, and the cleavage of PKC was determined by western blotting. The 40 kDa cleaved form is marked by an arrow. Results were reproducible, and blots shown are representative of three separate experiments. CL, cleaved form; FL, full-length form; HC, heavy chain.

View larger version (34K): [in a new window]   Fig. 3. Inhibition of PKC activity suppresses glutamate-induced ERK1/2 activation. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for 7 hours in the presence or absence of 5 µM rottlerin (Rott). (i) Phosphorylation of ERK1/2 in whole cell extracts was examined by western blot analysis using a phospho-ERK1/2 (pERK1/2)-specific antibody, and (ii) PKC activity was determined by immune complex kinase assay as described in the Materials and Methods. Results were reproducible, and blots shown are representative of three separate experiments. (B) HT22 cells were transfected with either a mock vector or a dominant-negative mutant (DN) of PKC (PKC DN) vector. After 12 hours, the cells were incubated with or without 5 mM glutamate for 7 hours. (i) Phosphorylation of ERK1/2 was examined by western blot analysis using a phospho-ERK1/2-specific antibody, and (ii) PKC activity was determined by immune complex kinase assay. Relative phospho-ERK1/2 levels shown in the columns below were determined from densitometric scanning of enhanced chemiluminescence-exposed film. Each bar is a mean ± s.d. value of three independent experiments normalized by arbitrarily setting the mock-transfected cell densitometrics to 1. (C) (i) Representative photomicrographs of apoptotic cells treated with or without 5 mM glutamate for 12 hours in the presence or absence of 5 µM rottlerin. (ii) HT22 cells were transfected with either control or PKC siRNA (siPKC) and, after 24 hours, cells were exposed to 5 mM glutamate for 9 hours. Downregulation of PKC after transfection was confirmed by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). Each bar is a mean ± s.d. value from three separate experiments. *P<0.05, **P<0.01.

View larger version (33K): [in a new window]   Fig. 4. MKP-1 regulates ERK1/2 phosphorylation during glutamate-induced oxidative toxicity. (A) HT22 cells were incubated with 5 mM glutamate (Glu) for the indicated times. The cells were then extracted to determine protein levels of MKP-1 by western blot analysis. Western blot analyses with anti-actin antibody in the same extracts are shown in the bottom panels. The results are from a representative experiment out of four separate experiments. (B) Cells were transfected with 2 µg of Myc-tagged vectors containing either wild-type MKP-1 or its catalytically inactive mutant (MKP-1CS), expression was allowed for 12 hours, and then cells were exposed to 5 mM glutamate for 9 hours. Whole cell extracts were subjected to western blot analysis using specific antibodies against phospho-ERK1/2 (pERK1/2), ERK1/2 and MKP-1. (C) HT22 cells were transfected with scrambled control (Scr) or MKP-1 siRNA (siMKP-1 73). (i) The level of MKP-1 at 24 hours after transfection was monitored by western blot analysis. The levels of phospho-ERK1/2, ERK1/2, PKC and GAPDH were also examined by western blot analysis. (ii) Phosphorylation of ERK1/2 under glutamate exposure for 7 hours was determined by western blot analysis. Relative phospho-ERK1/2 levels were determined by densitometric scanning of enhanced chemiluminescence-exposed film, and the levels were calculated by averaging the results obtained from three independent experiments. (D) Cells were transfected with either control (scrambled, Scr) or MKP-1 siRNAs [siMKP-1 73 (si 73) and siMKP-1 853 (si 853)]. After 24 hours, cells were treated with 5 mM glutamate for 7-9 hours, followed by western blot analysis. *P<0.05, **P<0.01.

View larger version (30K): [in a new window]   Fig. 5. Inhibition of PKC activity prevents glutamate-induced downregulation of MKP-1. HT22 cells were transfected with either a mock vector or a dominant-negative mutant (DN) of PKC (PKC DN) vector. After 12 hours, cells were exposed to 5 mM glutamate (Glu) for 7 hours. Western blot analysis was performed using specific antibodies against MKP-1 and actin. The relative MKP-1 protein levels were calculated by averaging the results obtained from three independent experiments. *P<0.05, ns, not significant.

View larger version (25K): [in a new window]   Fig. 6. PKC activation by glutamate induces ubiquitylation and downregulation of MKP-1 in HT22 cells. (A) HT22 cells were incubated with or without 5 µM ALLN or 5 µM lactacystine for 7 hours in the presence or absence of 5 mM glutamate (Glu). Western blot analysis was performed using specific antibodies against MKP-1 and actin. Relative MKP-1 protein levels were calculated by averaging the results obtained from three independent experiments. (B and C) Cells were treated with 5 mM glutamate in the presence or absence of 5 µM rottlerin (Rott) for 4 hours, followed by addition of 5 µM MG132 (MG) for 3 hours. Cells were then harvested, and subjected to immunoprecipitation (IP) of MKP-1 using anti-MKP-1 antibody. Following SDS-PAGE, membranes were immunoblotted (IB) with an anti-ubiquitin (anti-Ub) antibody (upper panel). The level of MKP-1 was determined by western blot analysis of the same cell lysates (lower panel). Results were reproducible, and blots shown are representative of three separate experiments.

View larger version (35K): [in a new window]   Fig. 7. Inhibition of PKC prevents ubiquitylation and degradation of MKP-1 in primary immature cortical neurons. (A) (i) Immature primary cortical neuron cultures prepared from day E13 mouse fetuses were seeded onto 24-well plates and grown for 24 hours. Cells were then incubated with or without 5 mM glutamate (Glu) in the presence or absence of 20 µM U0126 or 3 µM rottlerin (Rott) for 24 hours as indicated. Cell viability was analyzed by MTT assay. Results are mean ± s.d. from three separate experiments. **P<0.01 compared with control cells exposed to glutamate alone. (ii) Representative photomicrographs of apoptotic cells incubated with or without 5 mM glutamate in the presence or absence of 20 µM U0126 or 3 µM rottlerin for 24 hours, detected by TUNEL staining. (B) Cells were transfected with either control or PKC siRNA (siPKC) and, after 24 hours, cells were exposed to 5 mM glutamate for 16 hours. Downregulation of PKC was confirmed by quantitative RT-PCR (ii), and cell viability was measured by MTT assay (i). Each bar is mean ± s.d. value from three separate experiments. **P<0.01. (C) Cells were incubated with or without 5 mM glutamate in the presence or absence of 20 µM U0126 or 3 µM rottlerin for 12 hours. Cells were then harvested, lysed and phospho-(p)ERK1/2 or total ERK1/2 in equivalent amounts of total lysate protein (40 µg) were visualized by western blot analysis using specific antibodies against phospho-ERK1/2, ERK1/2 and actin. (D) Cells were incubated with or without 5 mM glutamate in the presence or absence of 5 µM rottlerin for 14 hours, followed by addition of 5 µM MG132 (MG) for 4 hours. Equal amounts of proteins in each cell extract were subjected to immunoprecipitation (IP) using 2 µg of anti-MKP-1 antibody. The immunoprecipitates were subjected to immunoblotting (IB) using antibody against ubiquitin (anti-Ub; upper panel). The level of MKP-1 was determined by western blot analysis of the same cell lysates (lower panel). Results were reproducible, and blots shown are representative of two independent experiments.

View larger version (29K): [in a new window]   Fig. 8. Role of MKP-1 in the regulation of glutamate-induced oxidative toxicity. (A) Exogenous expression of MKP-1 increases cell viability during oxidative toxicity. Cells were transfected with 2 µg of Myc-tagged vectors containing either wild-type MKP-1 or mock. After 12 hours, cells were exposed to 5 mM glutamate (Glu) for 9 hours. MKP-1 protein levels were determined by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). Results are mean ± s.d. obtained from three separate experiments. **P<0.01. (B) HT22 cells were transfected with control (Scrambled) or MKP-1 siRNA (siMKP-1 73) as indicated, expression was allowed for 24 hours, and then cells were exposed to 5 mM glutamate for 7 hours. Downregulation of MKP-1 after transfection was confirmed by western blot analysis (right panel) and cell viability was measured by MTT assay (left panel). Results are mean ± s.d. obtained from three separate experiments. (C) Immature primary cortical neuron cultures prepared from day E13 mouse fetuses were seeded onto 48-well plates and grown for 24 hours. (i) Cells were transfected with control or MKP-1 siRNA (siMKP-1 73; si 73) as indicated, expression was allowed for 24 hours, and then cells were exposed to 5 mM glutamate for 16 hours. Downregulation of MKP-1 after transfection was confirmed by western blot analysis (right panel), and cell viability was measured by MTT assay (left panel). (ii) Phosphorylation of ERK1/2 under glutamate exposure for 12 hours was determined by western blot analysis. Relative phospho-ERK1/2 (pERK1/2) levels were determined by densitometric scanning of enhanced chemiluminescence-exposed film, and the levels were calculated by averaging the results obtained from three independent experiments. (D) HT22 cells were transfected with either control or MKP-1 siRNA and, after 24 hours, cells were exposed to 5 mM glutamate in the presence or absence of 20 µM U0126 for 7 hours. Cell viability was measured by MTT assay. Results are mean ± s.d. obtained from three separate experiments. *P<0.05, **P<0.01.