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

DNA Binding Domain-independent Pathways Are Involved in EWS/FLI1-mediated Oncogenesis

Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in ∼85% of Ewing’s family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing’s tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways

The PHD and kinase domains are essential for MEKK1-dependent JNK activation.

<p><i>A</i>. Expression of mutations in relation to endogenous MEKK1. HA-His-tagged pBabeMEKK1 was introduced into <i>MEKK1^−/−</i> (−/−) DT40 cells by retroviral infection (reconstituted cells are referred to here as M-^Wt and MEKK1^Wt in the text), selected with puromycin and protein levels were assessed by western blot probed with αMEKK1 (left panel). The size difference between His-HA-tagged and endogenous MEKK1 is indicated. Each mutation was introduced into <i>MEKK1</i>^−/− cells and expression was compared (right panel). Reconstituted cell lines are referred to as M-^mutation within each figure. <i>B</i>. The PHD and kinase domain are required for c-Jun phosphorylation after vinblastine stimulation. Reconstituted cell lines were stimulated with 1 µM vinblastine (Vin) for four hours and lysed in modified RIPA. Phosphorylated c-Jun, total c-Jun and β-tubulin were assessed by western blot. <i>C</i>. There is a decreased amount of c-Jun phosphorylation in the <i>MEKK1</i>^−/−, PHD and kinase domain mutant cell lines after cytochalasin D treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 2 µg/ml cytochalasin D (Cy) treatment. <i>D</i>. There is a decreased amount of c-Jun phosphorylation in the <i>MEKK1^−/−</i>, PHD and kinase domain mutant cell lines after okadaic acid treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 90 nM okadaic acid (OA) treatment.</p

MEKK1^mutP is not phosphorylated in the activation loop after vinblastine treatment.

<p><i>MEKK1^−/−</i>, MEKK1^Wt, MEKK1^mutP or MEKK1^mutK (left panel) and MEKK1^delU, MEKK1^mutC (right panel) cell lines were treated with 1 µM vinblastine for 2 hours. Cell lysates were immunoprecipitated with αHA-agarose, run on SDS-PAGE gel and probed with αphospho-MEKK1 followed by detection of total MEKK1 with αMEKK1 C-22.</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

The PHD and kinase domains of MEKK1 are not essential for maximal ERK activation.

<p><i>A</i>. ERK is not activated or degraded after vinblastine treatment. Wild type, knockout and MEKK1-reconstituted <i>MEKK1^−/−</i> cells were treated with 1 µM vinblastine for four hours and total ERK or β-tubulin were assessed by western blot. <i>B</i>. Activation of ERK by cytochalasin treatment is not MEKK1-dependent. Cell lines were treated with 2 µg/ml cytochalasin D for two hours and phosphorylated and total ERK were evaluated by western blot. <i>C</i>. Activation of ERK by okadaic acid treatment is not MEKK1-dependent. Phosphorylation of ERK and total ERK were determined by western blot after treatment with 90 nM okadaic acid for four hours. β-tubulin was used as a loading control.</p

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

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