Research Summary
Kevan Shokat has developed chemical methods to decipher the role of individual kinases and their cellular signaling networks. His goals are to understand each kinase's role in the body and to learn which kinases should be targeted to treat diseases such as cancer and immune dysfunction.
Research in my laboratory is focused on using the tools of synthetic organic chemistry, structural biology, genetics, and mathematical modeling to gain insight into how signaling networks transmit information in normal and disease settings. We use chemistry to answer questions that cannot be addressed by the use of biochemistry or genetics—in this way we seek to provide tools that fill in gaps left behind by more traditional approaches. Our multidisciplinary work involves many collaborations with cell and organismal biologists, systems biologists, structural biologists, and computational biologists.
Chemical Tools for Studying the Function of Each Cellular Kinase
The cell uses a relatively limited repertoire of chemical modifications to orchestrate remarkable transformations. Transfer of phosphate from ATP to proteins and lipid second messengers are central to almost all signaling pathways and are catalyzed by more than 600 distinct enzymes to more than 10,000 or more cellular molecules. An understanding of signaling networks requires robust methods for deciphering the substrates of all phosphotransferases and the kinases responsible for each phosphoprotein. The utility of phosphorylation in biological systems is intimately connected with the reversible nature of the modification, and yet the phosphatase that counterbalances each kinase or the kinetics of the cycling of a given phosphate is almost completely unknown. We have developed chemical-genetic methods to address each of these challenges in kinase signaling.
Movie 1: Starting with the full domain structure of c-Src, the catalytic domain (blue) is shown, with the residues in the ATP-binding pocket (highlighted in yellow) surrounding ATP (shown as stick models). The remaining protein-protein interaction domains are shown in orange and purple. As the movie zooms into the active site, a cutaway through the surface of the protein shows the substrate ATP-binding pocket and surface of the c-Src kinase. Superimposed upon ATP is PP1, an inhibitor of the wild-type kinase. This molecule, a general inhibitor of many tyrosine kinases, fits into the active site analogously to how ATP binds.
As ATP disappears from view, the surface of the gatekeeper residue (T338 in c-Src) is highlighted in red to draw attention to the close contact with the phenyl ring of PP1. Next, an analog of PP1, 1-NAPP1, appears and makes direct contact (pokes through) the surface of the protein produced by the gatekeeper T338 residue. The PP1 (wild-type inhibitor) is removed, and the movie zooms in closer to the steric clash of 1-NAPP1 with the gatekeeper residue (red). The mutation of the gatekeeper residue to glycine (T338G) is introduced next, which allows 1-NAPP1 unimpeded access to the mutant kinase active site. The surface produced by Gly338 is much deeper and smaller than the Thr338 surface of the wild-type protein, providing the basis for exclusive selectivity for mutant protein kinases with a glycine at the gatekeeper position.
Finally the movie "rocks" to allow a three-dimensional impression of the features of the active site. The final frame of the movie represents how 1-NAPP1 encounters the conserved large gatekeeper residue in all wild-type kinases, providing exclusive specificity for engineered kinases containing an enlarged ATP-binding site.
Daniel Rauh, UCSF, HHMI postdoctoral fellow
Phosphoproteomics: Affinity Tags for Protein Kinase Substrates
The search for the complete set of all protein kinase substrates has become a major goal of many laboratories. It is estimated that one-third of the proteome is phosphorylated, making the tracing of the substrates of more than 500 kinases challenging. To address this problem, we have devised a chemical method for radioisotope tagging of the direct substrates of any protein kinase, using a [γ-32P]-labeled ATP analog, N6-(benzyl)ATP. This ATP analog is a poor substrate of wild-type protein kinases but is efficiently accepted by any kinase of interest by virtue of a mutation that enlarges the ATP-binding site to accommodate the N6-benzyl substituent.
We have developed additional chemical handles to capture the phosphate transferred by a single kinase. The key to kinase-catalyzed delivery of a phosphate affinity handle is to assemble the tag in two steps. First, a uniquely reactive phosphate mimic, phosphorothiolate (PO3S–), is delivered to kinase substrates via N6-(benzyl)ATP-γ-S and a mutant kinase. Next, a synthetic thiol-reactive electrophile is used to functionalize the phosphorothiolate and provide an affinity handle for antibody recognition. The antibody-based recognition is useful for immunoprecipitation of the direct substrates of a single kinase, as well as in-cell immunofluorescence of substrate localization. Proteomic identification of modified phosphate-containing proteins can also be accomplished with purely chemical means, which facilitates large-scale substrate identifications. The ability to directly affinity purify substrates of any kinase in the genome will allow for the mapping of any kinase pathway in a cell and development of a complete picture of the complex networks of kinase signal transduction pathways. Our long-term goal is to use these chemical tools to identify all the direct substrates of each kinase in the human genome. We are also developing approaches for introducing these ATP analogs directly into intact cells, allowing for the direct labeling of substrates in their undisturbed cellular compartments.
Protein Kinase Inhibitors: Toward a Pharmacological Map of Cell Signaling
A central experimental paradigm used for probing components of signal transduction pathways is perturbation through induced loss of function. Biochemical approaches are often limited because signaling networks span from the cell surface to the control of transcription and translation, confounding reconstitution efforts from purified proteins. Genetic approaches allow perturbation of single components in an intact cell or organism, yet they are often confounded by the rapid evolvability of the networks. Chemical and pharmacological approaches enable rapid, reversible, and graded (dose-dependent) inactivation of single components in intact cells or organisms. Unfortunately, highly selective chemical probes (e.g., agonists, antagonists, traceable substrates) of protein kinases are difficult to develop because the 500 protein kinases share highly homologous ATP-binding pockets.
To solve this fundamental problem in the case of protein kinases, we have developed a strategy that combines protein engineering and organic synthesis. This approach, which we have termed chemical genetics, relies on genetics to specify the target of a small molecule, ensuring that only the intended protein is targeted by the small molecule we synthesize. Through mutation of a large conserved residue, the "gatekeeper residue," in the ATP-binding pocket of kinases to a nonnatural small amino acid (glycine), we can sensitize any kinase to inhibition by 1-NAPP1, which only inhibits kinases containing a glycine at the gatekeeper position. (See the short animation, based on the crystal structure of the tyrosine kinase c-Src, which highlights the structural basis for selectivity of 1-NAPP1 and the mutation of the gatekeeper position that allows inhibition by 1-NAPP1.) (This mutation is identical to that described above for allowing the kinase to use N6-(benzyl)ATP as an orthogonal substrate.)
The chemical-genetic approach for generation of monospecific inhibitors of any protein kinase is a powerful method for mapping cell signaling. Toward our goal of developing a specific small-molecule inhibitor of every protein kinase in the human, mouse, yeast, worm, and fly genomes, we have applied the chemical-genetic approach to more than 75 protein kinases. These studies have revealed novel and unexpected aspects of kinases involved in cell cycle control, growth factor receptor activation, map kinases, antiapoptosis kinases, neuronal kinases, plant receptor kinases, and pathogen-specific kinases.
Kinases Control Network Feedback to Fine-Tune Responses
A recently emerging aspect of kinase cascades is the presence of feedback regulation. Since feedback loops are used to control input-output of pathways on a rapid timescale, pharmacological methods are ideal for studying pathway dynamics. Most importantly, feedback has been shown to be the mechanism used by cancer cells to generate resistance to kinase inhibitors. In some cases, the feedback induced by drugs causes a remarkable and dangerous acceleration of cancer growth. Several anticancer drug trials have been stopped as a result of these effects. We have focused on three aspects of feedback control. (1) how to short-circuit feedback by targeting multiple steps in a pathway—an approach we term "polypharmacology"; (2) identification of the precise molecular basis for feedback control (e.g., surprisingly, inhibitors can "hijack" kinase activation by regulating kinase localization); (3) single-cell measurements and mathematical modeling, which may reveal the consequences of pathway architecture on output sensitivity and noise resistance.
Chemical Methods for Studying Chromatin
Protein methylation is a particularly critical form of regulation involved in chromatin structure. Since a given histone can be modified at numerous lysine residues with numerous patterns of between one and three methyl groups per lysine, it has been difficult to determine the role of a single methyl group on complex chromatin structure. We have developed new chemical methods for synthesis of large quantities of homogeneously methylated histones of any pattern. These chemically modified histones have been useful for many laboratories and have been incorporated into single nucleosomes and larger nucleosome arrays to test chromatin-modifying enzyme activities. They have also been used to solve the first x-ray structure of a nucleosome containing a methylated lysine histone.
A grant from the National Institutes of Health provided partial support for the work on phosphoproteomics.
As of May 30, 2012
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