Signal Transduction

M. Muzio, A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz, M. Mann, P. H. Krammer, M. E. Peter, V.M. Dixit, "FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex," Cell, 85:817-27, 1996. (Cited more than 420 times since publication) Comments by Vishva M. Dixit, Director of Molecular Oncology, Genentech Inc., South San Francisco, Calif. OPENING DOORS: A technique Genentech's Vishva Dixit and colleagues used to identify parts of the cell-death signaling complex has since aided research in other fields. Some components of the cell's intricate death effector machinery have eluded discovery. Characterizing these signaling complexes has been a major goal for researchers studying signal transduction. But many of these signaling proteins are simply present in quantities too small to be subjected to protein sequence analysis. Earlier analytical methods either failed to detect the signaling molecules or yielded false positives. More recently, a technique developed by Matthias Mann, head of the European Molecular Biology Laboratory, and adapted by Vishva M. Dixit, then at the University of Michigan at Ann Arbor, and Peter Krammer, investigator at the German Cancer Research Center in Heidelberg, expanded the analytical arsenal. Mann's technique--nano-electrospray tandem mass spectrometry (nano-ES-tandem MS)--allows proteins to be sequenced with greater sensitivity than earlier methods. Dixit notes: "Mann's innovation was to invent an analytical tool where he could get sequence information from exceedingly small amounts of protein." As little as 5 nanograms of a protein isolated by simple polyacrylamide gel electrophoresis can be partially sequenced using this technique. A signaling complex can be examined by isolating it, resolving its constituent components by gel electrophoresis, and identifying individual components using a combination of nano-ES-tandem MS and database searching. Screening databases with partial sequences often reveals the identity of the encoding gene. The technique was first applied, by Mann, to E. coli (M. Wilm et al., Nature, 379:466-9, 1996). Dixit, Mann, and Krammer were the first group to use this technique to identify a mammalian protein. The results are featured in this paper. Another group reached similar conclusions, reported in the same issue of Cell and in an earlier Hot Paper, by employing the yeast two-hybrid procedure instead (The Scientist, 12[3]:13, Feb. 2, 1998). Both papers identified components of the Fas/APO-1 death-inducing complex that bound to the receptor-associated adaptor molecule FADD, although each paper named them differently. Dixit and colleagues termed the protein FLICE, while the other paper called it MACH. The accepted name is now caspase-8. This caspase plays a key role in apoptosis. After binding to FADD, caspase-8 becomes activated. It then triggers a signaling cascade that leads to cell death. "Since FLICE is a death protease, its recruitment and subsequent activation by a death receptor was a pivotal step in understanding how a death signal is transduced from a cell surface receptor into the cell's interior," Dixit says. However, Dixit adds that the methodology leading to the caspase's discovery may be as important as the discovery itself. The technique opens up research in other fields, including signaling, transduction, and DNA repair, Dixit notes: "We can now sequence and identify proteins that were previously unidentifiable." Since the publication of this paper, other labs have adopted the nano-tech procedure to solve some of the most intractable problems in cellular signaling. These include the identification of the I * B kinase (F. Mercurio et al., Science, 278:860-6, 1997), analysis of the complex that catalyses V(D)J recombination (U. Grawunder et al, Nature,388:492-5, 1997), and cloning of the elusive and highly sought telomerase enzyme (J. Lingner et al., Science, 276:561-7, 1997). As far as Dixit's aware, only a few labs in the United States are using the technique. He suspects the paper's high number of citations mean that eventually more labs will adopt the technique: "It's only a matter of time before it becomes widely used."

Signal Transduction

The Scientist Staff

Interested in reading more?

Become a Member of