Melissa Dix, a research associate in Cravatt's laboratory,and Gabriel Simon, a then-graduate student now working as a postdoctoralresearcher 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 cleavageevents in cells during a cellular process of interest. To build on it, theresearchers add a technique for identifying phosphorylation events, as well asSILAC, a recently developed proteomics technology that allows researchers todistinguish, within given samples, copies of proteins that hail from differentcell populations.
The combined technique was applied to populations of controland apoptotic cells, and resulted in the detection of more than 700apoptosis-specific proteolysis events (most mediate by apoptosis-drivingenzymes known as caspases), including several that had not yet been reported.The mapping also revealed a large, apoptosis-specific series of phosphorylationevents, 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 wasthe other way around. Additionally, some kinase enzymes that phosphorylateproteins during apoptosis were found to be incapable of carrying out theirduties 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 effectiveinhibitors of caspases, development of selective inhibitors, that target only one specific caspase, has proven to be challenging."
Cravatt's laboratory is now applying the techniqueselsewhere, initially in studies of apoptosis in a variety of cell types. It isthought that the ability to detect apoptosis in specific types of cells couldhave applications in cancer diagnostics and therapies, given that tumor cellsgenerally evolve resistance to apoptosis. The research team is seeking todiscover if certain phosphorylated protein fragments could be used asbiomarkers of apoptosis in cancer cells, allowing researchers to gauge theeffectiveness 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 beyondapoptosis.
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 thepaper, "Functional interplay between caspase cleavage and phosphorylationsculpts the apoptotic proteome," in the July 20 edition of
Cell. Additional co-authors include Chu Wang and Eric Okerberg, ofCravatt's lab, and Matthew P. Patricelli, now of ActivX Biosciences, Inc. inSan Diego.
The study received funding from the National Institutes ofHealth, the California Breast Cancer Foundation,
ActivX Biosciences, the
ARCSFoundation and the Skaggs Institute for Chemical Biology at Scripps Research.
