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Talking with cells
LA JOLLA, Calif.óCell communication is a familiar aspects for biologists and researchers, given the functions of mRNA and the fact that many therapeutics are based on taking advantage of or interrupting the interaction between proteins and receptors. But some of the latest research to come out of the Scripps Research Institute has revealed that even cellular events such as phosphorylation and proteolysis coordinate during cellular processes.
Phosphorylation is the attachment of a phosphate group to a protein, and proteolysis is the cleavage of a protein, and both processes are mediated by enzymes, occurring after a protein has been translated and folded. Some phosphorylation and proteolysis events activate proteins to take their places in cellular processes, while others deactivate proteins. Up until now, the two forms of protein modification have generally been studied separately, but the new approach, which combines techniques to map the events across all proteins in a cell population, has revealed that the processes cooperate to carry out apoptosis.
"Detecting the cross-talk between protein regulation pathways has long been a challenge, and so with this new technique we can start to do analyses that were difficult or impossible before," said senior investigator Benjamin F. Cravatt, professor and chair of the Department of Chemical Physiology at Scripps Research and member of Scripps Research's Skaggs Institute for Chemical Biology.
Previous studies have suggested that phosphorylation and proteolysis work in tandem occasionally, but the studies had focused on specific apoptosis-driving enzymes and their biochemical partners, rather than on the "global" apoptosis process.
Melissa Dix, a research associate in Cravatt's laboratory, and Gabriel Simon, a then-graduate student now working as a postdoctoral researcher at Washington University, St. Louis, also took part in the study. The pair built on a previous proteolysis-mapping method, known as PROTOMAP, that they had described in 2008 in the journal Cell. PROTOMAP can generate detailed pictures of protein cleavage events in cells during a cellular process of interest. To build on it, the researchers add a technique for identifying phosphorylation events, as well as SILAC, a recently developed proteomics technology that allows researchers to distinguish, within given samples, copies of proteins that hail from different cell populations.
The combined technique was applied to populations of control and apoptotic cells, and resulted in the detection of more than 700 apoptosis- specific proteolysis events (most mediate by apoptosis-driving enzymes known as caspases), including several that had not yet been reported. The mapping also revealed a large, apoptosis-specific series of phosphorylation events, several of which evinced clear connections to proteolysis events.
"Our recent research elucidated an extensive network of bidirectional cross-talk between kinases and caspases, specifically that phosphorylation within caspase cleavage sites can promote caspase-mediated proteolysis, and caspase cleavage can directly promote phosphorylation--both mechanisms, to our knowledge, have not been previously reported," says Dix.
Dix and Simon determined that in some cases, phosphorylations enabled the caspase cleavage events, while in others, it was the other way around. Additionally, some kinase enzymes that phosphorylate proteins during apoptosis were found to be incapable of carrying out their duties unless they were cleavage-activated by caspases.
"Caspases have long been thought of as attractive drug targets," says Dix. "These proteases have very defined cleavage sequence specificity. While this specificity has proven useful in designing very effective inhibitors of caspases, development of selective inhibitors, that target only one specific caspase, has proven to be challenging."
Cravatt's laboratory is now applying the techniques elsewhere, initially in studies of apoptosis in a variety of cell types. It is thought that the ability to detect apoptosis in specific types of cells could have applications in cancer diagnostics and therapies, given that tumor cells generally evolve resistance to apoptosis. The research team is seeking to discover if certain phosphorylated protein fragments could be used as biomarkers of apoptosis in cancer cells, allowing researchers to gauge the effectiveness of cancer treatments by way of standard blood tests. Dix notes that while this was originally studied in cancer, the method could have potential in identifying cross-talk in other areas where pathogenesis is driven by proteolysis and phosphorylation. In addition, the new technique is also capable of mapping other cellular processes beyond apoptosis.
Dix says that moving forward, the team will follow up on "some of the more interesting targets we identified in this study, specifically the protein fragments that are phosphorylated following caspase proteolysis." The researchers will also be looking to expand the platform to examine cross-talk between "other post-translational pathways, such as ubiquitination and acetylation," Dix notes.
The results of the Scripps team's work appeared in the paper, "Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome," in the July 20 edition of Cell. Additional co-authors include Chu Wang and Eric Okerberg, of Cravatt's lab, and Matthew P. Patricelli, now of ActivX Biosciences, Inc. in San Diego.