BOSTON — Even yeast understand austerity. A finely tuned system evolved early on to help cells survive in a world where good times come as fast as they go. The system, a molecular switch found in organisms from yeast to humans, involves a nutrient-sensing protein that turns growth on in times of plenty and shuts it off when times are lean.
New work from the lab of Wenyi Wei, PhD, an investigator in the Department of Pathology at Beth Israel Deaconess Medical Center and J. Wade Harper, PhD, of the Department of Cell Biology at Harvard Medical School (HMS) has uncovered the mechanism that flips this switch. They found that mTOR, the mammalian version of this key protein, turns itself on in a positive feedback loop involving destruction of DEPTOR, the protein that holds the mTOR switch in the off position. The results are reported in the October 20 online edition of Molecular Cell along with two other papers reporting similar findings.
The mTOR pathway has been linked to cancer in cases where an abnormality in the signaling allows cells to grow uncontrollably regardless of fitness, so complete understanding of the intricacies of mTOR signaling could contribute to the design of novel cancer treatments. The pathway has also been linked to cardiovascular, autoimmune and metabolic diseases. This new work answers key questions about how the switch flips but also raises intriguing new questions about how an inactivated protein can activate itself.
When Wei, Assistant Professor of Pathology at HMS, began this investigation, parts of the mTOR switch mechanism had already been uncovered. For instance, DEPTOR holds mTOR in the off position by binding with it and blocking its activity. Further, DEPTOR vanishes through ubiquitination, a process that tags proteins for destruction and sends them to the proteasome, a disposal system that shreds proteins when they are no longer needed.
But the molecular mechanism that triggers DEPTOR to vanish was unknown. Wei, an expert in ubiquitination, decided to take on the problem.
To find out which protein(s) signals DEPTOR to degrade, the investigators employed a technology called ComPASS developed by Harper, the Bert and Natalie Vallee Professor of Molecular Pathology at HMS. Developed in 2009, ComPASS specifically identifies protein interactions and protein complexes.
In the case of DEPTOR, Wei and Harper found that the E3 ligase beta-TrCP marks DEPTOR for disposal by tagging it with ubiquitins. This tagging causes DEPTOR to be rapidly depleted, unleashing mTOR and, in turn, cell growth.
But beta-TrCP only tags DEPTOR if it has been phosphorylated, a process that marks a protein with phosphates to activate or deactivate it. So, asked Wei, how does DEPTOR get phosphorylated?
In a screening process to identify the proteins responsible for phosphorylating—and therefore depleting—DEPTOR, he found two culprits: casein kinase 1 (CK1) and mTOR. “We were really surprised to find mTOR itself degrading DEPTOR,” says Wei. “In a starvation state, DEPTOR levels are high and mTOR should not be able to do anything.”
But when nutrients come in, something changes. With the deftness of Houdini, mTOR escapes from DEPTOR’s binds and springs into action. The first thing mTOR does is join with CK1, a helper protein kinase that is always waiting around on standby. Together, mTOR and CK1 phosphorylate DEPTOR, triggering a rapid ubiquitination response from beta-TrCP and the immediate degradation of DEPTOR.
It is this mysterious feat that has now has Wei’s attention. “How can mTOR be activated when it is fully suppressed by DEPTOR? We really want to know that,” he says. The team is actively examining this question.
While this signaling pathway originally evolved as a nutrient sensor, in more complex organisms it acts as a fitness sensor. “All sorts of stimuli are integrated into one circuit,” says Bing Su, Associate Professor of Immunobiology at Yale University School of Medicine. “This particular study provides one piece of the puzzle to help us understand this complicated signaling process. However, there are many pieces of the puzzles remaining to be found.”
Aberrations in the mTOR signaling pathway have been implicated in cancer, cardiovascular disease, autoimmunity and metabolic disorders. They have also been identified in a phenomenon called the Warburg Effect, which allows cells to grow not only in nutrient-starved conditions but also in the oxygen-deprived environment typically found inside tumors.
A drug called rapamycin, which inhibits mTOR (in fact, its name is an acronym for Mammalian Target of Rapamycin), is being studied as a potential targeted drug for many forms of cancer. But the drug only acts against one of the forms of mTOR, which appears in two complexes: mTORC1 and mTORC2. “A drug that could inhibit both would be better,” says Kun-Liang Guan, professor of pharmacology at the University of California at San Diego. “This research found how to get rid of DEPTOR, and maybe provided a way to keep DEPTOR, which would inhibit both.”
Designing drugs that target mTOR’s complex signaling pathway requires a deep understanding of the subtle molecular interactions that naturally govern the system so that pharmaceutical interventions tug in the right places at the right times. “The more you understand the whole,” says Wei, “the better you can design drugs to alter it.”
This work was supported in part by the Department of Defense, the National Institutes of Health, the Lady Tata Memorial Trust, and A-STAR. Harper is a consultant for Millennium Pharmaceuticals.
Coauthors include BIDMC investigators Hiroyuki Inuzuka (co-first author), Jason Locasale, Pengda Liu, Lixin Wan, Rebecca Chin, Shavali Shaik, Costas Lyssiotis, Alex Toker,, Lewis Cantley, and John Asara; and Havard Medical School investigators Marcus Tan (co-first author), Bo Zhai and Steven Gygi.
BIDMC Contact: Bonnie Prescott
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