Master Cell Controller

For this article, Eugene Russo interviewed Yosef Shiloh, a professor of human genetics at Tel Aviv University's Sackler School of Medicine, and Michael Kastan, chairman, department of hematology-oncology at St. Jude Children's Research Hospital in Memphis, Tenn. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. S. Banin, L. Moyal, S.Y. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, "Enhanced phosphorylation of p53 by ATM in response to DNA damage," Science, 281:1674-7, Sept. 11, 1998. (Cited in more than 210 papers since publication) C.E. Canman, D.S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, J.D. Siliciano, "Activation of the ATM kinase by ionizing radiation and phosphorylation of p53," Science, 281:1677-9, Sept. 11, 1998. (Cited in more than 210 papers since publication) People often ask Yosef Shiloh, a professor of human genetics at Tel Aviv University's Sackler School of Medicine, why the focus of his life's work is ataxia telangiectasia (A-T), an extremely rare autosomal recessive disorder. His answer is twofold: Firstly, he says, patients and families with very rare diseases are as entitled as anyone to the attention and the effort of the biomedical community. Secondly, despite A-T's scarcity, a better understanding of the disease, the mutated gene causing A-T, ATM (ataxia telangiectasia-mutated), and its protein product could actually have wide-ranging ramifications in several areas of biomedical research. Because A-T is characterized by such varied symptoms, including neuronal degeneration, premature aging, immune deficiency, acute radiation sensitivity, and increased cancer risk, understanding the disorder could provide insight into the particulars of biomedical pathways key to fields like neurology, oncology, and immunology. These two papers helped toward that end by characterizing the kinase activity of ATM and demonstrating its phosphorylation of the oft-studied and frequently mutated tumor suppressor gene p53. "The question has always been, how can one protein account for all these defects and control so many functions?" explains Shiloh. His group cloned ATM in 1995,1 work that itself has been cited in more than 870 subsequent publications. "And it turns out that basically ATM controls so many functions simply by phosphorylating key players in the pertinent pathways." P53, as reported in these papers, was the first of several ATM targets identified. In 1991, researchers that included Michael Kastan, chairman of the department of hematology-oncology at St. Jude Children's Research Hospital in Memphis, Tenn., showed that DNA damage induced p53.2 The following year they showed that p53 induction, after exposure to DNA damage-triggering ionizing radiation, was abnormal in cells from patients with A-T.3 "What that told us was that whatever that gene product was, it was upstream of p53 in that signaling pathway," Kastan comments. The next major advance didn't come until Shiloh's 1995 cloning of ATM, findings that suggested ATM had a kinase motif. Follow-up research demonstrated that p53 is phosphorylated in response to DNA damage on serine 15, a particular site in the amino terminal domain, and that ATM was important for the phosphorylation event. "So then the natural question that arose was, since ATM has a kinase motif, is it in fact the kinase that's directly phosphorylating p53?" explains Kastan. According to these two papers, it was. Both groups developed in vitro assays for measuring the kinase activity of ATM. The assays allowed them to demonstrate that ATM was a bona fide protein kinase and to map the site in p53 that ATM phosphorylates, namely serine 15, the same site already shown in vivo to phosphorylate in p53 in response to DNA damage. Kastan and Shiloh, who met at a meeting about seven years ago and began regularly exchanging data, had only minor methodological differences in their approaches--approaches that effectively complemented one another. Shiloh's lab used endogenous ATM for a kinase assay; Kastan's used cDNA, which, because it is easily manipulated, made it easier to confirm that ATM was a bona fide protein kinase. Also, Shiloh's lab used the radiomimetic drug neocarzinostatin (NCS) rather than ionizing radiation to induce DNA damage; by using NCS, Shiloh was able to confirm that a double DNA strand break, and not some other effect of ionizing radiation, had activated ATM. Michael Kastan "Getting the kinase assay to work led to many, many studies that were relying on this assay basically to look for additional ATM substrates and additional pathways controlled by ATM in this manner," Shiloh remarks. "The number of ATM substrates is growing quite rapidly." Kastan, Shiloh, and others continue to look for additional ATM substrates, some of which could be feasible drug targets. Using the p53 target sequence as a starting point, Kastan's group actually has identified a general consensus target sequence for ATM that has helped elucidate other proteins, and sites in those proteins, that get phosphorylated by ATM. ATM substrates implicated thus far include the breast cancer susceptibility gene BRCA1, a protein called CtIP that binds to BRCA1, the cell cycle checkpoint protein Chk2, the p53 regulator MDM2, and a protein called p95Nbs1 (nibrin) that has been implicated in Nijmegen breakage syndrome (NBS). Phosphorylations by ATM may either activate or inactivate these target proteins. The ATM assay has helped researchers to understand how these signaling pathways are involved in radiation-induced carcinogenesis and to identify potential ways to manipulate radiation responses for therapeutic benefit. "Just sorting through these radiation signaling pathways is important for understanding tumor development and tumor responses to therapy," says Kastan. Shiloh's group is currently doing an epidemiological study on the ongoing controversial issue of the possible predisposition of A-T heterozygous individuals to cancer. They're also using microarrays to study gene expression in normal versus A-T cells. As ATM-related research, spurred in large part by these two papers, continues to proliferate, scientists continue to decipher what Shiloh labels a "master controller of cellular responses." Eugene Russo can be contacted at erusso@the-scientist.com References 1. K. Savitsky et al., "A single ataxia-telangiectasia gene with a product similar to PI-3 kinase," Science, 268:1749-53, 1995. 2. M.B. Kastan et al., "Participation of p53 protein in the cellular-response to DNA damage," Cancer Research, 51:6304-11, 1991. 3. M.B. Kastan et al., "A mammalian-cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia," Cell, 71:587-97, 1992. 4. D.S. Lim et al., "ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway," Nature, 404:613, April 6, 2000.

Master Cell Controller

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