Aug. 26, 2003 A chemical sleight of hand by UCSF scientists has pinpointed for the first time where small molecules called phosphates bind to proteins in cells, allowing them to send signals and giving organisms a way to adapt to rapidly changing conditions.
Diabetes, hypertension and many cancers arise when these simple molecules bind incorrectly or not at all. Mapping their location on proteins should allow researchers to block aberrant binding and treat disease, the scientists say.
The addition of phosphates is nature's dominant way of transferring information within and between cells, the researchers say. One third of all proteins in the human body carry phosphates; some proteins are studded with as many as 50 of the small molecular add-ons. In essence, the phosphate addition, known as phosphorylation, induces a protein to send a signal to a second protein. Such signals are vital for survival, allowing cells to respond to changes in their environment by, for example, regulating the production and transport of hormones, nutrients, and the like.
But the common signaling system is vulnerable to sabotage. Many cancer cells rely on aberrant phosphorylation to avoid a normal death, either by carrying more phosphate bonds than normal or making excess proteins with the normal number of phosphates, the scientists say. The opposite can also be life-threatening: Insufficient phosphorylation can cause diseases such as diabetes by dampening the cellular effects of insulin.
"This finding will allow us for the first time to quickly identify all the changes in protein phosphorylation that occur in a cell," said Kevan Shokat, PhD, professor of cellular and molecular pharmacology at UCSF and also professor of chemistry at UC Berkeley. "Since phosphorylation controls so many biological processes and is involved in so many diseases, mapping the sites where it takes place will identify new therapeutic targets."
Shokat is senior author on a paper reporting the research in the September issue of Nature Biotechnology. The paper was published on-line this week.
Lead author is Zachary Knight, a UCSF graduate student in chemistry and chemical biology who conceived of the way to trace the precise bonding sites of phosphates on proteins by using use a type of selective chemistry. Last year, Knight won the $20,000 Grand Prize in the annual Collegiate Inventors Competition for this discovery. Shokat was awarded $10,000 for his role.
Shokat, Knight and UCSF have applied for a patent covering the new chemical approach to map phosphates – the first such success. The research was carried out in Shokat's laboratory at UCSF's new Mission Bay campus.
As nearly ubiquitous as it is, the process of adding phosphates to protein is not part of the genetic master plan. The modification occurs after the protein has been faithfully produced according to instructions in the gene. A molecule known as a protein kinase ferries a phosphate-laden molecule to a specific amino acid along the chain that makes up a protein. The addition of the phosphate – simply composed of one phosphorus atom and four oxygen atoms – changes the chemical nature of the amino acid, which in turn changes the protein's shape and prompts a new interaction with a second protein.
"Every cellular process is controlled at some level by protein phosphorylation," Knight said. "It is the language by which cells communicate and make decisions."
Until now, researchers could identify which proteins had phosphate bonds, but they could not easily determine where they were. By knowing bond locations, scientists should be able to correlate specific phosphate bonding patterns with specific diseases, Shokat said. Drugs could then be designed to block the specific version of protein kinase that normally carries phosphate to that specific site. There are more than 600 different protein kinases in humans, each one committed to delivering phosphates to unique amino acid sites on proteins.
Over the past 15 years, scientists have been able to map the structure of many proteins by cutting them in various places with a protease – most often trypsin -- and then using the tool of mass spectrometry to measure the mass of molecular fragments that result. But phosphates' high energy bonds make them unstable and difficult to measure; they break off from proteins so readily that it is difficult to use mass spectrometry to determine their original locations.
Shokat and Knight solved this problem by figuring out a way to transform the protein so that the protease -- rather than the mass spectrometer -- can tell them the location of the phosphate groups. Because no proteases are known that recognize phosphate groups, this type of approach was not believed to be possible before their work. But Knight devised a chemical transformation that sheds phosphates from an amino acid and converts the molecule into one that looks like lysine -- the favored amino acid target for trypsin. In essence, this conversion tricks the protease into cutting at a site that it ordinarily would not recognize, and in doing so, pinpoints the original location of the phosphate group.
The Shokat lab carried out the chemical sleight of hand, while a collaborator, Bradford Gibson, PhD, and his colleagues performed all the mass spectrometry analysis.
Gibson is a researcher at the Buck Institute for Age Research and an adjunct professor of pharmaceutical chemistry at UCSF.
Co-authors on the paper are Brigit Schilling, PhD, a post-doctoral fellow, and Richard Row, a research technician, at the Buck Institute; and Denise Kenski, a graduate student in Shokat's lab.
The research was funded by the National Institutes of Health.
The Collegiate Inventors Competition that Knight won is hosted by the National Inventors Hall of Fame, rewarding inventions by graduate students in the natural sciences.
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