Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1

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

Characterization of MAPK activation in wild type and MEKK1-deficient cell lines.

<p><i>A</i>. Phosphorylation of c-Jun phosphorylation is defective in the <i>MEKK1</i>^−/− chicken DT40 cells in response to cytoskeletal disruption. Wild type (Wt) and <i>MEKK1</i>^−/− DT40 cells were left untreated (U) or stimulated with 1 µM vinblastine (Vin), 2 µg/ml cytochalasin D (Cy), and 90 nM okadaic acid (OA) for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel to compare wild type and knockout cell lines. Western blot membranes were probed for phosphorylated and total c-Jun, ERK and p38. <i>B</i>. Phosphorylation of c-Jun is defective in <i>MEKK1</i>^−/− murine pre-B cells in response to cytoskeletal disruption. Wild type (Wt) and <i>MEKK1</i>^−/− pre-B murine cells were stimulated with 1 µM vinblastine, 2 µg/ml cytochalasin D, and 90 nM okadaic acid for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel. Membranes were probed for phosphorylated and total c-Jun, ERK and p38. β-tubulin is used as loading control.</p

The PHD and kinase domains are required for vinblastine induced apoptosis.

<p><i>A</i>. <i>MEKK1^−/−</i> DT40 cells reconstituted with MEKK1 or mutations thereof all initiate apoptosis in response to etoposide. Each reconstituted cell line was treated with 25 µM etoposide (Et) for four hours and subjected to DNA laddering assay. <i>B</i>. <i>MEKK1^−/−</i> cell lines reconstituted with MEKK1 containing the PHD or kinase mutations do not result in DNA fragmentation after vinblastine treatment. Each reconstituted cell line was treated with 1 µM vinblastine (Vin) for six hours and subjected to a DNA laddering assay. <i>C</i>. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after cytochalasin D treatment. Reconstituted cell lines were treated with 2 µg/ml cytochalasin D (Cy) for six hours and subjected to DNA laddering assays. <i>D</i>. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after okadaic acid treatment. Reconstituted cell lines were treated with 90 nM okadaic acid (OA) for six hours and subjected to DNA laddering assays.</p

Identifying the post-translational modifications of the MEKK1 protein.

<p><i>A</i>. The PHD mutant can be phosphorylated. Vector, wild type flag-MEKK1 and flag-PHD mutant (mutP) were transfected into 293T cells and immunoprecipitated with αflag M2 conjugated beads. Membranes were probed with total or phospho-MEKK1. <i>B</i>. CIP treatment does not affect basal MEKK1 modification, but does shift the molecular weight of vinblastine treated (phosphorylated) endogenous MEKK1. DT40 cells were treated with vinblastine for six hours and lysates were immunoprecipitated with αMEKK1. Half of each lysate was treated with calf alkaline phosphatase and all were incubated at 37°, run on SDS-PAGE gel and membranes were probed with αMEKK1. <i>C</i>. Inhibition of de-ubiquitinating enzymes via N-ethylmaleimide (NEM) stabilizes the higher molecular weight form of MEKK1. Wild type DT40 cells were lysed in modified RIPA. Lysates were treated with or without 20 mM NEM for 30 minutes at room temperature. Lysates were run on SDS-PAGE and probed with α MEKK1. <i>D</i>. MEKK1 is ubiquitinated whereas the PHD mutant is not. 293T cells were transiently transfected with vector, flag-MEKK1 or mutP. Cell lysates were immunoprecipitated with flag-conjugated beads, run on an SDS-PAGE gel and probed with αubiquitin or αMEKK1.</p

Quantification of apoptosis by propidium iodide incorporation and caspase 3 activity.

<p><i>A</i>. In the MEKK1^mutP and MEKK1^mutK cell lines the percentage of apoptotic cells does not significantly increase after cytoskeletal disruption. To quantify the levels of DNA fragmentation, propidium iodide staining was analyzed by flow cytometry. Cell lines were treated with 1 µM vinblastine (panel I), 2 µg/ml cytochalasin D (panel II) or 90 nM okadaic acid (panel III) for twelve hours and the percentage of apoptotic cells were determined by measuring fluorescence lower than the diploid population using CellQuest software. <i>B</i>. Caspase 3 is not activated in the MEKK1^mutP or MEKK1^mutK cell lines after cytoskeletal disruption. Caspase 3 activity was determined after four hours of treatment with 1 µM vinblastine (I), 2 µg/ml cytochalasin D (II) or 90 nM okadaic acid (III) treatment. Cell lysates were used to determine the catalytic activity of caspase 3 by using the colorimetric substrate Ac-DEVD-pNA and measured at 405 nM.</p

Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines.

<p><i>A</i>. DNA laddering is significantly decreased in the <i>MEKK1^−/−</i> 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 <i>MEKK1^−/−</i> 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 <i>MEKK1−/−</i> murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. <i>B</i>. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or <i>MEKK1^−/−</i> 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 <i>MEKK1^−/−</i> 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. <i>C</i>. Caspase 3 activation is abrogated in the <i>MEKK1</i>^−/− cell lines after cytoskeletal disruption. DT40 and <i>MEKK1^−/−</i> 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 <i>MEKK1^−/−</i> 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.</p

Creation and characterization of MEKK1 mutations.

<p><i>A</i>. Schematic diagram of MEKK1, and MEKK1 mutants illustrating the plant homeodomain (MutP), the ubiquitin-interacting motif (delU), the kinase domain (MutK) and the putative caspase cleavage site (MutC). Arrows indicate the mutations created within each domain. <i>B</i>. Proper protein expression and determination of downstream signaling via c-Jun phosphorylation after overexpression of each mutation. Each mutant was transiently transfected into 293T cells in the highly expressing pApuro construct. Cell lysates were run on SDS-PAGE, western blots were performed and MEKK1 was detected with αMEKK1 C-22. Blots were next probed with total c-Jun, which recognizes both the dephosphorylated and phosphorylated forms of c-Jun. <i>C</i>. Cellular localization of each mutant. HeLa cells were transfected with pApuro MEKK1, MutP, delU, MutK or MutC and stained with DAPI (nucleus) and αMEKK1 C-22 antibody (MEKK1) after 24 hours of transfection. <i>D</i>. The PHD mutant does not colocalize with lysosomes to a greater extent than wild type MEKK1. HeLa cells were transfected with flag-MEKK1 or flag-MutP and stained with DAPI, M2 αflag (MEKK1) and LAMP1 (lysosomes) after 24 hours. MEKK1 was detected with mouse αFITC and LAMP1 with rabbit αTRITC. <i>E</i>. The PHD mutant localization is not altered by vinblastine treatment. HeLa cells were transfected with pApuro MEKK1, MutP and MutK. After 24 hours cells were treated with vinblastine for one hour. MEKK1 was visualized with αMEKK1 C-22 antibody and nuclei visualized with DAPI.</p