One of the most important classes of human enzymes are protein kinases – ; Signaling molecules that regulate nearly all cellular activities, including growth, cell division, and metabolism. Dysfunction in these cellular pathways can lead to a variety of diseases, particularly cancer.
Identification of protein enzymes involved in cellular dysfunction and cancer development could lead to many new drug targets, but for the vast majority of these kinases, scientists do not have a clear picture of the cellular pathways they are involved in, or what their substrates are.
“We have a lot of sequencing data for cancer genomes, but what we’re missing is the large-scale study of the signaling pathway and the activation states of protein kinases in cancer. If we had that information, we’d have a much better idea of how it works,” says Michael Yaffe, David H. Koch Professor of Science at MIT. Technology, director of the MIT Center for Precision Cancer Medicine, a member of the Koch Institute for Integrative Cancer Research, and a senior author on the new study.
Yaffe and other researchers have created a comprehensive atlas of more than 300 protein enzymes found in human cells, and identified the proteins they are likely to target and control. This information can help scientists decipher many cell signaling pathways, and help them discover what happens to those pathways when cells become cancerous or are treated with certain drugs.
Louis Cantlie, professor of cell biology at Harvard Medical School and the Dana-Farber Cancer Institute, and Benjamin Turk, assistant professor of pharmacology at Yale School of Medicine, are also senior authors on the paper, which appears today in nature. The lead authors of this paper are Jared Johnson, instructor of pharmacology at Weill Cornell Medical College, and Tomer Yaron, graduate student at Weill Cornell Medical College.
The human genome includes more than 500 protein kinases, which activate or deactivate other proteins by binding them to a chemical modification known as a phosphate group. For most of these kinases, the proteins they target are unknown, although research on kinases such as MEK and RAF, which are involved in cellular pathways that control growth, has led to new cancer drugs that inhibit those kinases.
To identify additional dysregulated pathways in cancer cells, researchers rely on phosphoproteins using mass spectrometry-; A technique that separates particles based on their mass and charge – ; To detect proteins that are highly phosphorylated in cancer cells or healthy cells. However, to date, there has been no easy way to interrogate mass spectrometry data to determine which protein kinases are responsible for the phosphorylation of those proteins. Because of this, it has remained unknown how these proteins are regulated or misregulated in disease.
“For most of the phosphopeptides that are measured, we don’t know where they fit in the signaling pathway. We don’t have a Rosetta stone that you can use to look at these peptides and say, ‘This is the pathway the data is telling us about,'” Yaffe says. “The reason for this is that for most protein enzymes, we don’t know what their substrates are.”
Twenty-five years ago, while a postdoctoral researcher in Cantlie’s lab, Yaffe began studying the role of protein kinases in signaling pathways. Turk joined the lab soon after, and spent all three decades studying these enzymes in research groups of their own.
“This is a collaboration that began when Ben and I were in Lou’s lab 25 years ago, and now it’s finally coming together, driven in large part by what the lead authors, Jared and Tomer, have done,” Yaffe says.
In this study, the researchers analyzed two classes of kinases -; serine kinases and threonine kinases, which make up about 85 percent of the protein kinases in the human body -; based on what kind of structural form they put the phosphate groups on.
By working with a library of peptides that Cantley and Turk had previously created to search for motifs with which the kinases interact, the researchers measured how the peptides interacted with all 303 known serine and threonine enzymes. Using a computational model to analyze the interactions they observed, the researchers were able to identify the kinases capable of phosphorylating Each of the 90,000 known phosphorylation sites reported in human cells, for these two classes of kinases.
To their surprise, the researchers found that several kinases with very different amino acid sequences had evolved to bind and phosphorylate the same forms on their substrates. They also showed that about half of the movements they studied target one of three major categories of motifs, while the remaining half are specific to one of about a dozen smaller categories.
The new kinase atlas can help researchers identify signaling pathways that differ between cancerous and normal cells, or between treated and untreated cancer cells, Yaffe says.
“The atlas of kinase morphotypes now allows us to decode signaling networks,” he says. “We can look at all those phosphorylated peptides, and we can put them back on a specific kinase.”
To prove this approach, the researchers analyzed cells treated with an anticancer drug that inhibits a kinase called Plk1, which regulates cell division. When they analyzed the expression of the phosphorylated proteins, they found that many of those affected were controlled by Plk1, as they expected. To their surprise, they also discovered that this treatment increased the activity of two kinases involved in the cellular response to DNA damage.
Yaffe’s lab is now interested in using this atlas to try to find other ineffective signaling pathways that lead to cancer development, particularly in certain cancers for which no genetic drivers have been found.
“We can now use phosphoproteins to say, maybe in this patient’s tumor, these pathways are up-regulated or these pathways are down-regulated,” he says. “It has the potential to identify signaling pathways that lead to cancer in circumstances where it is not clear what genes drive cancer.”
The research was funded by the Leukemia & Lymphoma Society, the National Institutes of Health, Cancer Research UK, the Charles and Marjorie Holloway Foundation, the MIT Center for Precision Cancer Medicine, and a Koch Institute (core) support grant. From the National Cancer Institute.