Johns Hopkins Medicine
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Media Contact: Joanna Downer
December 16, 2004
SURPRISE! CELLS HAVE SECOND SOURCE OF PHOSPHATE
New Source Means New Cellular Communication
For 50 years, thousands of labs around the world have studied cells' critical internal communications, and scientists had assumed the speakers were known. But now, in the Dec. 17 issue of Science, Johns Hopkins researchers report finding not just a new participant, but a brand new conversation that has implications for treating disease and understanding biology.
Much of cells' internal communication revolves around two very important words -- "stop" and "go" -- elicited when a small bit, called phosphate, is added onto proteins. This addition turns protein activities up or down and fine tunes cells' responses to what's happening outside their borders. This communication can go awry in diseases, including cancer, and be corrected by various drugs.
The source of these phosphate bits has been known -- a molecule called adenosine triphosphate, or ATP. But in their new report, the Johns Hopkins scientists describe a brand new source of phosphate that seems to work with as many proteins as targeted by ATP, but in a completely different way.
"There are already drugs that affect particular roles of ATP to treat cancer and other conditions, so we envision drugs that increase or decrease specific activities of this new source of phosphate could be important in neurologic and psychiatric illnesses, and perhaps in cancer as well," says Solomon Snyder, M.D., professor and director of neuroscience, one of the departments in Johns Hopkins' Institute for Basic Biomedical Sciences.
"Nobody in a million years would have thought there was another way for cells to add phosphate groups to proteins other than using ATP," he adds. "Addition of phosphates to proteins -- phosphorylation -- is the most fundamental signaling mechanism in all life, and the new source of phosphate represents a very different kind of process than the one we've known about. It represents a totally new form of cellular communication."
Unlike ATP, the new phosphate source, known as inositol pyrophosphate (IP7), modifies proteins without any help, just binding directly to the protein and leaving behind one of its phosphates, the researchers report. Their early evidence also suggests IP7 might be most important in regulating the release of chemicals in the brain and in controlling the cellular machinery that builds proteins.
While IP7's newly found role is likely to surprise many, Snyder has been expecting it. In the early 1990s, he noticed the first reports that IP7 and a related molecule called IP8 existed, interesting to him because for 15 years, he'd been studying related inositol (pronounced in-AH-si-tahl) phosphates.
But unlike the molecules he'd been working with, which look like bracelets with three to six phosphate "charms," IP7 and IP8 had too many phosphates to fit on the bracelet. Instead, the seventh and eighth phosphates would have to be linked to another phosphate "charm" rather than to the "bracelet" itself.
"ATP has a similar phosphate-phosphate connection, so I speculated that IP7 and IP8 might also be able to give up that extra phosphate," says Snyder, who is also a professor of pharmacology and molecular sciences and of psychiatry. "Proving it turned out to be very difficult technically."
First, it took several years for then-graduate student Susan Voglmaier to isolate an enzyme that builds IP7 (from IP6), an advance the team published in 1996. It took a few more years for postdoctoral fellow Adolfo Saiardi, Ph.D., to clone the three enzymes that make IP7 and to use them to make IP7 in which the extra phosphate was radioactive.
After finally making sufficient quantities of radioactive IP7, Saiardi and postdoctoral fellow Rashna Bhandari, Ph.D., mixed it with a "puree" of mouse brain or kidney, which produced hundreds of radioactive proteins. After numerous experiments to rule out other possibilities, the researchers could finally conclude that IP7 gives away its extra phosphate to proteins.
"We think IP7 phosphorylation of proteins is as universal as ATP phosphorylation," says Snyder, whose lab is continuing to study IP7's protein targets and where on proteins its phosphates are added. "The enzymes that build IP7 are most prevalent in the brain, but they are found everywhere. What we've learned so far is just the tip of the iceberg."
Already, Bhandari has discovered that two of the proteins most heavily phosphorylated by IP7 are involved in the ribosome, the cellular machine that reads genetic instructions and constructs proteins. IP7 also controls the cellular "mail room" -- the preparation and release of tiny packages that contain messengers, such as neurotransmitters in the brain that create movement, memory and mania.
The researchers also have shown in work described in the Journal of Biological Chemistry, online now, that proper activity of one of the enzymes that makes IP7 is critical in cell death -- because of IP7's role in modifying proteins.
"So drugs that activate the enzyme and stimulate production of IP7 would increase cell death, which is what one wants to do in cancer treatment," says Snyder. "Drugs inhibiting the enzyme would prevent cell death, the goal in treating stroke and neurodegenerative diseases."
The first proof of how ATP works was the impetus for the 1992 Nobel Prize in Physiology or Medicine. During the mid-1950s, the awardees, Americans Edmond Fischer and Edwin Krebs, isolated the first example of an enzyme that takes ATP's phosphate and gives it to a protein, and showed that the phosphate changes the protein's function.
Since then, scientists have found and studied thousands of other ATP-controlled proteins. While IP7 also controls protein activity, its role in cellular communication -- coordinating internal activities to respond to external events -- is likely quite distinct from ATP's, given the differences Snyder and his colleagues have already observed. IP7 may even add its phosphate to phosphates already on proteins, which, if confirmed, is completely unheard of, says Snyder.
Authors are Saiardi, Bhandari, Snyder, Adam Resnick and Adele Snowman, all of Hopkins. Saiardi is now at University College London. The research was funded by the National Institute of Mental Health and the National Institute on Drug Abuse.
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