11 months ago

2004 Scientific Report

Seals, Darren F., and

Seals, Darren F., and Sara A. Courtneidge. 2003. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes and Development 17(1): 7–30. Voytyuk, Olexandr, Johan Lennartsson, Akira Mogi, Georgina Caruana, Sara Courtneidge, Leonie K. Ashman, and Lars Ronnstrand. 2003. Src family kinases are involved in the differential signaling from two splice forms of c-Kit. Journal of Biological Chemistry 278(11): 9159–9166. From left to right, back: Bromann, Pass, Freiter, Azucena, Uzarski, Seals front row: Salinsky, Cruz, Tesfay, Korkaya 20

Laboratory of Developmental Cell Biology Nicholas S. Duesbery, Ph.D. Dr. Duesbery received both his M.S. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research Development Center, Maryland. Laboratory Members Staff Xudong Liang, M.D. John Young, M.Sc. Research Interests Our research group uses biochemical and molecular approaches to elucidate regulatory mechanisms involved in human health and disease and in the early embryonic development of Xenopus species. One of our main projects is described in the following text. The lethal effects of Bacillus anthracis have been attributed to an exotoxin it produces that is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA binds to a cell surface receptor and, upon proteolytic activation to a 63-kDa fragment, forms a heptameric membrane channel that mediates the entry of three molecules of LF or EF into the cell. EF is an adenylate cyclase that together with PA forms a toxin referred to as edema toxin. LF is a Zn 2+ -metalloprotease that together with PA forms a toxin referred to as lethal toxin (LeTx). LeTx is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals. To date, mitogen-activated protein kinase kinase (MEK) molecules are the only identified physiological substrates of LF. A comparison of the LF cleavage sites on MEKs 1–4, 6, and 7 reveals elements of homology. In all cases the cleavage site is preceded by a series of basic residues and followed immediately by an aliphatic residue. Also, with the exception of MEKs 3 and 4, the cleavage site is preceded by one or more proline residues. Synthesizing these results, the consensus site for LF cleavage fits the pattern B·B/P·B·P·(X) 2–3·Al, where B represents a basic residue; P, proline; X, variable; and Al, aliphatic residue. This motif is similar to one in a described generic mitogen-activated protein kinase (MAPK) binding site, or docking (D) domain, consisting of a basic amino acid center flanked by hydrophobic residues on one or both sides. This raises the possibility that LF may be a D-domain protease that targets both activators and substrates of MAPKs. However, using in vitro cleavage assays, we have been unable to detect LF-induced cleavage of CL-100, c-jun, or ATF-2, known substrates of MAPKs that contain a D domain. Thus, other regions of MEKs, in addition to the NH 2 -terminal cleavage site, must be required for LF substrate recognition. Using mutational analysis, we have identified a functionally conserved COOH-terminal region of MEKs that is essential for LF-mediated proteolysis of MEK. The presence of a conserved region distal to the cleavage site, which is necessary for binding and/or cleavage by LF, may explain in part the failure to identify physiological LF substrates other than MEKs. The fact that other MEK1-regulatory proteins (such as B-Raf, PAK, and the scaffolding protein MP-1) also interact with MEK in this region suggests that the region constitutes a key regulatory domain of MEK1 and perhaps of other MEKs. This possibility may have functional implications for LF toxicity, since the presence of MEK-binding proteins may alter LF’s access to its substrates. Conversely, LF might decrease MEK activity by competitively displacing positive regulators of MEKs. However, by itself this mechanism seems insufficient to explain how LF inactivates MEKs, because we have observed that LF can inhibit the activity of constitutively 21

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