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2004 Scientific Report

activated MEK1. Since

activated MEK1. Since phosphorylation under these conditions depends upon not only kinase activity but also substrate affinity, we reasoned that LF might inhibit MEK by either reducing its intrinsic kinase activity or decreasing its affinity for ERK. The latter seemed more likely given that MEK1 deletion mutants lacking the 32 NH 2 - terminal residues are deficient in their ability to bind ERK and that mutations in the docking domain decrease the efficiency with which MEK1 activates ERK. However, we have found that as well as decreasing MEK’s affinity for its substrate, LF also decreases MEK’s intrinsic kinase activity. The latter result was not expected but is not without precedent. Based on homology to the A-helix of cAMP-dependent protein kinase and the observation that NH 2 -terminal deletions and activation lip substitutions synergize to activate MEK1, Ahn and colleagues have hypothesized that regions of the NH 2 terminus form long-range interactions with the activation loop and that perturbation of the structure within this region promotes conformational changes in the loop that favor activation. By analogy, we predict that the NH 2 terminus of MEK1 associates with its activation loop to promote its activity. The NH 2 -terminal structure may also promote protein stability, because we have noted that the long-term stability of MEKs is decreased in cells treated with PA and LF. The proximity of the D domain to the activation loop may coordinate MEK-ERK interaction and facilitate ERK phosphorylation and activation. Increased MEK activity has been associated with many aspects of tumorigenesis. The combined roles of multiple MEK pathways in tumorigenesis and the unique activity of LF in inhibiting each of these pathways suggests that LeTx may be an effective therapeutic agent for the treatment of cancer. Indeed, preliminary screening of LeTx against the National Cancer Figure 1. Lethal toxin inhibits growth of colorectal adenocarcinomas. The WiDr human colorectal adenocarcinoma cell line was used to generate xenograft tumors in nude mice. Control mice were treated with buffered saline only (representative tumor, left). A tumor from a mouse that received lethal toxin every second day by tail vein injection is shown at right. Institute’s 60–cell line anti-neoplastic drug screen indicates that several tumor types, including melanomas and colorectal adenocarcinomas, are sensitive to growth inhibition by lethal toxin. We have shown that LeTx can inhibit the growth and vascularization of murine fibrosarcomas and, in collaboration with Han-Mo Koo’s group at VARI, we have demonstrated that human melanoma-derived cell lines undergo apoptosis following treatment with LeTx. We have continued this work, assessing the efficacy of LeTx against human colorectal carcinoma–derived cell lines in vitro as well as in vivo. Whereas LeTx in vitro induced cell cycle arrest and apoptosis of the HT-29 and WiDr cell lines, respectively, it had no effect upon proliferation of the SW-620 cell line. By contrast, the pathological malignancy and growth of each of these cell lines as xenografts in athymic nude mice was significantly reduced following intravenous treatment with LeTx (Fig. 1). Our results indicate that proteolytic inhibition of multiple MEK signaling pathways reduces the growth and malignancy of human colorectal adenocarcinoma. 22

External Collaborators Stephen Leppla, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Robert Liddington, Burnham Institute, La Jolla, California Angel Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany Art Frankel, Wake Forest University, Winston-Salem, North Carolina Jean-François Bodart, Université des sciences et technologies de Lille, France. Recent Publications Singh, Y., X. Liang, et al. In press. Pathogenesis of Bacillus anthracis: the role of anthrax toxins. Microbial Toxins. T. Proft., ed. Norfolk, U.K.: Horizon Scientific. Chopra, Arun P., Sherrie A. Boone, Xudong Liang, and Nicholas S. Duesbery. 2003. Anthrax lethal factor proteolysis and inactivation of MAPK kinase. Journal of Biological Chemistry 278(11): 9402–9406. Frankel, Arthur E., Han-Mo Koo, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2003. Novel protein-targeted therapy of metastatic melanoma. Current Pharmaceutical Design 9(25): 2060–2066. Perdiguero, Eusebio, Marie-Jeanne Pillaire, Jean-Francois Bodart, Florian Hennersdorf, Morten Frödin, Nicholas S. Duesbery, Gema Alonso, and Angel R. Nebreda. 2003. Xp38γ/SAPK3 promotes meiotic G2/M transition in Xenopus oocytes and activates Cdc25C. EMBO Journal 22(21): 5746–5756. Frankel, Arthur E., Bayard L. Powell, Nicholas S. Duesbery, George F. Vande Woude, and Stephan H. Leppla. 2002. Anthrax fusion protein therapy of cancer. Current Protein and Peptide Science 3(4): 399–407. From left to right: Duesbery, Young, Liang 23

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