Cytoplasmic FRAP must enter and exit nucleus to perform

December 18, 2000

CHAMPAIGN, Ill. -- University of Illinois biologists have discovered that a protein that lives in the cytoplasmic world between a mammalian cell's membrane and nucleus undergoes a "nuclear experience" that is necessary for regulating cell growth and division. Coupled with a similar finding involving a different protein in yeast cells, announced in the journal Cell in 1999 by Harvard Medical School researchers, the new understanding of how the protein-synthesis machinery is regulated in living cells could have significant implications in biology.

"What we found is a brand new mechanism for cell signaling, a fundamental one that was not realized until it was shown in yeast," said Jie Chen, a UI professor of cell and structural biology. "Our research is the first to show this activity in mammalian systems." Chen and UI graduate student Jae Eun Kim found that the FKBP12-rapamycin-associated protein (FRAP) regulates a membrane-to-cytoplasm communication network - called signal transduction - that governs cellular response to outside stimuli such as growth factors, hormones or foreign agents. If FRAP fails to briefly enter and exit the nucleus then some crucial proteins fail to synthesize, thereby preventing cell growth and proliferation.

"This is important because of the link to human diseases, especially cancer, which are basically cases of cell growth gone awry," she said. "What we have found is a regulatory mechanism for normal cell growth. If we understand normal cell growth better, we can come up with better ways to fight deregulated cell growth."

The findings of Chen and Kim were published in the Dec. 19 issue of the Proceedings of the National Academy of Sciences. The National Institutes of Health and American Heart Association funded the research.

Signal transduction occurs in the cytoplasm within a complex, spider-web-like network where chains of proteins and receptors intermingle. It is also where FRAP was long believed to dwell and perform its various functions. Researchers had seen the protein in the nucleus of mammalian cells, but its presence was thought to be an artifact of the experiments that were being conducted, Chen said. The idea that FRAP could get there on its own, considering its size and the previous notion of where it functioned, simply had been dismissed until new molecular technologies opened new research doors. A new possibility, Chen said, was that FRAP might be shuttling rapidly in and out of the nucleus.

Using cultured kidney cells from both humans and monkeys, UI researchers found that when they administered leptomycin B, a drug that blocks the export of material out of the nucleus, the protein was found naturally inside.

"Sure enough, we saw an accumulation of FRAP in the nucleus," Chen said. "It was so strong that it couldn't be an artifact. By taking away export from the equation, we saw the majority of the protein was getting into the nucleus. This was a very simple experiment, but it was very convincing."

Looking more closely at what happens when FRAP is not allowed to exit the nucleus led to a startling discovery. "We found to our surprise that the subsequent signal transduction events were blocked," she said. Next, Chen and Kim genetically engineered FRAP in a way that allowed them to manipulate the protein's ability to go into and out of the nucleus to test the impact on signaling within the cells.

"When we enhanced the protein export," Chen said, "we saw a decrease of signaling downstream in the network. And when we enhanced the protein import into the nucleus, we saw elevated signal transduction. The bottom line was that an increase of signaling was abolished when we blocked the export. FRAP's just going into the nucleus is not good enough; it has to come out to activate the pathway."

The findings strongly suggest that "you have to have a perfect balance between in and out," Chen said. "This protein has to shuttle between the nucleus and the cytoplasm to function. It has to have a nuclear experience to function in the cytoplasm."

The new mystery is just what happens during the brief time FRAP passes through the nucleus. "We think this nuclear shuttling allows for the coordination of the multiple roles FRAP may play in regulating pleiotropic [seemingly unrelated] cellular functions, which further underscores a central regulator role of FRAP," Chen said.

The Harvard finding involving yeast, in research led by Elaine A. Elion and published in August 1999, focused on the protein Ste5, a cytoplasmic protein that appears to act much like FRAP does in mammalian cells. Researchers there found that Ste5 was unable to activate a signal necessary for normal signal transduction unless it had gone in and out of the nucleus.

"Finding the same type of mechanism in both yeast and mammalian cells makes this new view of normal cell activity very convincing," Chen said. "The findings of these two studies are highly significant, because they suggest a new function of the nucleus in the regulation of signal transduction."

University of Illinois at Urbana-Champaign

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