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CELL BIOLOGY How Does Nucleus Tell Signals Apart? It's in the Timing Sensing Mechanism Points Toward New Cancer Targets The extracellular envoys lining up at the portals of the cell are likely to be a highly diverse bunch and, indeed, a wide variety of growth factors and hormones may rub shoulders with one another. Yet there is a reasonable chance that once inside the cell, their messages will be ferried to the nucleus by the more uniform mitogen-activated proteins, or MAP kinases. Which raises a conundrum: how do proteins inside the nucleus know whether a message is being sent by a growth factor versus a hormone if they are both delivered by the same MAP kinase pathway? Growth factors produce MAP kinase signals of varying duration. John Blenis and Leon Murphy (below, l to r) found that sustained (green) and transient (red) signals produce, in turn, a different response in the nucleus. Both activate genes such as fos, but only the sustained response causes the gene product to be phosphorylated. This phosphorylation stabilizes the protein and exposes a MAP kinase docking site, the DEF domain, paving the way for a second phosphorylation. The doubly phosphorylated protein activates genes necessary for cellular activities such as proliferation. (Illustration by Jeff Cleary; photo by Graham Ramsay) A report by John Blenis, Leon Murphy, and colleagues supports a hunch long held by researchers that the answer lies in the duration of the signals. The researchers found that the nuclear protein c-Fos, a well-known recipient of MAP kinase messages, is able to distinguish between long and short signals by means of an elegant mechanism. The findings appear in the August Nature Cell Biology. "This mechanism allows the cell to distinguish between subtle differences in timing, which may explain a lot of things we have not understood for many years in this field," said Blenis, HMS professor of cell biology. What is more, other nuclear proteins appear to utilize this same mechanism, raising the possibility that duration may be used by a wide variety of signaling pathways as a way of telling nuclear proteins how to respond to specific messages. "The ability of a cell to sense differences in signal timing tells us how a cell can utilize the same basic signaling machinery to do very different things like grow, die, differentiate into a specific kind of cell or, if improperly regulated, turn into a cancer cell," Blenis said. The Phosphate Two-step The mechanism depends, as do so many things in the cellular world, on the addition of phosphate groups--in this case, a two-step phosphorylation. The initial phosphorylation, which occurs only in the presence of a sustained, as opposed to a transient, MAP kinase signal, stabilizes c-Fos. At the same time, it causes a hidden stretch of amino acids to be revealed as a docking site for more MAP kinases, which phosphorylate c-Fos yet again. Only in this doubly phosphorylated state will c-Fos activate genes necessary for cell proliferation. "What stands out in this study is that the MAP kinases have been known to send a signal to c-Fos, and c-Fos is induced by many different stimuli," said Michael Greenberg, HMS professor of neurology at Children's Hospital, who is not an author on the study. "By showing differential phosphorylation of c-Fos in response to differential MAP kinase activation, one gets a sense of how a common transcriptional regulator might have somewhat different functions." The findings could also point to new methods for intervening when such signaling goes awry, as it does in cancer. Many tumors exhibit defects in the MAP kinase pathway, and especially in the ras gene, which ultimately result in sustained c-Fos signaling. One of the challenges has been to find a way to turn off c-Fos signaling while sparing the many MAP kinase signals that are critical to the cell's integrity. Targeting the c-Fos docking sites, thereby preventing c-Fos from undergoing its second phosphorylation, might be one approach. Murphy, an HMS research fellow in cell biology, is currently working with colleagues at the Institute of Chemistry and Cell Biology to develop screens for small molecules that will block c-Fos's docking sites. "We're interested in that predominantly for basic research purposes, but it is possible that anything we identify might also be used as a starting block to identify drugs that could be clinically useful," said Blenis. "That's the dream." Mapping the Target It is more than a passing fancy. Blenis, who helped define the role of Ras in the MAP kinase pathway, has been trying to find a way to characterize and target the MAP kinase pathway's signaling system since the early 1990s. He and his colleagues showed then that the MAP kinase pathway altered signal length depending on the growth factor that sent it. They also showed that sustained MAP kinase signals resulted in c-Fos phosphorylation. Building on this work, Murphy, who came to the Blenis lab three and a half years ago, discovered that sustained MAP kinase signaling activated not just the c-fos gene but also the c-Fos protein, while transient signaling only caused activation of the c-fos gene. Not only that, the c-Fos gene product produced by sustained signaling appeared to be more stable than that produced by transient signaling. Suspecting that this stabilization might be priming c-Fos for a second phosphorylation, Murphy began scouring the c-Fos protein for MAP kinase docking sites, including a newly identified one, the DEF domain. He found it. To his delight, c-Fos's DEF domain not only recruited MAP kinases, but it was located in the vicinity of phosphorylation sites. "That kind of fit very nicely," he said. The story was clinched when he found that fibroblasts with mutant c-Fos DEF domains would not divide. "So the docking sites appear to be really critical for c-Fos-mediated cell proliferation," said Murphy. In fact, the researchers have found DEF-like domains on the products of other immediate early genes, the proto-oncogenes c-myc and n-myc, and on a circadian clock gene product, mPer1. It is not clear what role it plays in these proteins. "What we are saying is that the DEF domain is likely to be pretty important in recruiting MAP kinases to these gene products," said Murphy. In fact, the docking site could serve several functions. "There may be other ways of utilizing the DEF domain," said Blenis. For example, it could serve to anchor proteins in the nucleus. "It might be a very nice way to tell proteins where to go to signal," he said. "The fact that it appears in such widespread pathways makes it important to really get at some of the details about it," said Greenberg. --Misia Landau