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Apoptosis induced by cytoskeletal disruption requires distinct domains of MEKK1.

Tricker E, Arvand A, Kwan R, Chen GY, Gallagher E, Cheng G - PLoS ONE (2011)

Bottom Line: MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug.We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption.Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

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Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines.A. DNA laddering is significantly decreased in the MEKK1−/− DT40 and murine pre-B cell lines upon treatment with vinblastine, cytochalasin D, and okadaic acid, whereas the response to etoposide is unchanged. Wild type (Wt) or MEKK1−/− DT40 cell lines were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), 90 nM okadaic acid (OA), or DMSO (DM) for six hours or 25 µM etoposide (Et) for four hours. After treatment, each line was assessed for the presence of DNA laddering. Wild type (Wt) or MEKK1−/− murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. B. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or MEKK1−/− DT40 were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 12 hours. Wild type (Wt) or MEKK1−/− murine pre-B cells were treated with 1 µM vinblastine (Vin), 4 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 14 hours. After treatment, the percentage of apoptotic cells was assessed by measuring fluorescence of the sub-diploid population using flow cytometry. C. Caspase 3 activation is abrogated in the MEKK1−/− cell lines after cytoskeletal disruption. DT40 and MEKK1−/− cells (left panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin for four hours and 90 nM okadaic acid for six hours. Murine wild type and MEKK1−/− pre-B cells (right panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin and 90 nM okadaic acid for six hours. Cell lysates were used to determine the catalytic activity of caspase 3.
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pone-0017310-g002: Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines.A. DNA laddering is significantly decreased in the MEKK1−/− DT40 and murine pre-B cell lines upon treatment with vinblastine, cytochalasin D, and okadaic acid, whereas the response to etoposide is unchanged. Wild type (Wt) or MEKK1−/− DT40 cell lines were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), 90 nM okadaic acid (OA), or DMSO (DM) for six hours or 25 µM etoposide (Et) for four hours. After treatment, each line was assessed for the presence of DNA laddering. Wild type (Wt) or MEKK1−/− murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. B. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or MEKK1−/− DT40 were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 12 hours. Wild type (Wt) or MEKK1−/− murine pre-B cells were treated with 1 µM vinblastine (Vin), 4 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 14 hours. After treatment, the percentage of apoptotic cells was assessed by measuring fluorescence of the sub-diploid population using flow cytometry. C. Caspase 3 activation is abrogated in the MEKK1−/− cell lines after cytoskeletal disruption. DT40 and MEKK1−/− cells (left panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin for four hours and 90 nM okadaic acid for six hours. Murine wild type and MEKK1−/− pre-B cells (right panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin and 90 nM okadaic acid for six hours. Cell lysates were used to determine the catalytic activity of caspase 3.

Mentions: Next, we investigated whether apoptosis induced by these drugs requires MEKK1. Strong DNA laddering was observed in the wild type DT40 and murine B cell lines in response to vinblastine, cytochalasin D and okadaic acid (Figure 2A). However, reduced levels of laddering were seen in both chicken and murine knockout cell lines. This effect did not appear to be a general defect in apoptosis, as treatment with the DNA damaging agent etoposide induced apoptosis in all cell lines (lane Et, Figure 2A). Apoptosis in response to each drug was then quantified by PI exclusion assay for each cell line (Figure 2B). In addition, we assessed caspase 3 activity to show that cells were undergoing apoptosis and not just DNA damage, and found that there is an increased level of active caspase 3 after each drug treatment in the wild type cells when compared to the knockout cells, which is consistent with the DNA laddering and PI exclusion data (Figure 2C). Taken together, these data indicate that MEKK1 is involved in both JNK activation and the apoptotic response to a number of cytoskeletal disrupting agents.


Apoptosis induced by cytoskeletal disruption requires distinct domains of MEKK1.

Tricker E, Arvand A, Kwan R, Chen GY, Gallagher E, Cheng G - PLoS ONE (2011)

Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines.A. DNA laddering is significantly decreased in the MEKK1−/− DT40 and murine pre-B cell lines upon treatment with vinblastine, cytochalasin D, and okadaic acid, whereas the response to etoposide is unchanged. Wild type (Wt) or MEKK1−/− DT40 cell lines were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), 90 nM okadaic acid (OA), or DMSO (DM) for six hours or 25 µM etoposide (Et) for four hours. After treatment, each line was assessed for the presence of DNA laddering. Wild type (Wt) or MEKK1−/− murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. B. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or MEKK1−/− DT40 were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 12 hours. Wild type (Wt) or MEKK1−/− murine pre-B cells were treated with 1 µM vinblastine (Vin), 4 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 14 hours. After treatment, the percentage of apoptotic cells was assessed by measuring fluorescence of the sub-diploid population using flow cytometry. C. Caspase 3 activation is abrogated in the MEKK1−/− cell lines after cytoskeletal disruption. DT40 and MEKK1−/− cells (left panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin for four hours and 90 nM okadaic acid for six hours. Murine wild type and MEKK1−/− pre-B cells (right panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin and 90 nM okadaic acid for six hours. Cell lysates were used to determine the catalytic activity of caspase 3.
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pone-0017310-g002: Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines.A. DNA laddering is significantly decreased in the MEKK1−/− DT40 and murine pre-B cell lines upon treatment with vinblastine, cytochalasin D, and okadaic acid, whereas the response to etoposide is unchanged. Wild type (Wt) or MEKK1−/− DT40 cell lines were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), 90 nM okadaic acid (OA), or DMSO (DM) for six hours or 25 µM etoposide (Et) for four hours. After treatment, each line was assessed for the presence of DNA laddering. Wild type (Wt) or MEKK1−/− murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. B. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or MEKK1−/− DT40 were treated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 12 hours. Wild type (Wt) or MEKK1−/− murine pre-B cells were treated with 1 µM vinblastine (Vin), 4 µg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 µM etoposide (Et) for 14 hours. After treatment, the percentage of apoptotic cells was assessed by measuring fluorescence of the sub-diploid population using flow cytometry. C. Caspase 3 activation is abrogated in the MEKK1−/− cell lines after cytoskeletal disruption. DT40 and MEKK1−/− cells (left panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin for four hours and 90 nM okadaic acid for six hours. Murine wild type and MEKK1−/− pre-B cells (right panel) were treated with 1 µM vinblastine and 2 µg/ml cytochalasin and 90 nM okadaic acid for six hours. Cell lysates were used to determine the catalytic activity of caspase 3.
Mentions: Next, we investigated whether apoptosis induced by these drugs requires MEKK1. Strong DNA laddering was observed in the wild type DT40 and murine B cell lines in response to vinblastine, cytochalasin D and okadaic acid (Figure 2A). However, reduced levels of laddering were seen in both chicken and murine knockout cell lines. This effect did not appear to be a general defect in apoptosis, as treatment with the DNA damaging agent etoposide induced apoptosis in all cell lines (lane Et, Figure 2A). Apoptosis in response to each drug was then quantified by PI exclusion assay for each cell line (Figure 2B). In addition, we assessed caspase 3 activity to show that cells were undergoing apoptosis and not just DNA damage, and found that there is an increased level of active caspase 3 after each drug treatment in the wild type cells when compared to the knockout cells, which is consistent with the DNA laddering and PI exclusion data (Figure 2C). Taken together, these data indicate that MEKK1 is involved in both JNK activation and the apoptotic response to a number of cytoskeletal disrupting agents.

Bottom Line: MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug.We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption.Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

Show MeSH