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Summary: Kevan Shokat has developed a chemical-genetics technique to decipher individual kinases and their cellular signaling networks. His goals are to understand each kinase's role in the body and to learn which kinases would be good drug targets.

Research in my laboratory is focused on the discovery of new chemical-based tools to decipher cellular signaling networks, with an emphasis on protein kinases. The analysis of signal transduction pathways is challenging using the traditional tools of biochemistry, genetics, and chemistry. 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.)

	QuickTime Movie: Engineering unique inhibitor sensitivity in a protein kinase...

We have also developed chemical-genetic tools for identification of novel direct kinase substrates of any protein kinase in the genome. In addition to inhibiting and/or tracing the function of any kinase in the genome, we have developed the first ATP-competitive agonist of a protein kinase, a chemical cross-linker capable of trapping phosphoproteins to the kinases responsible for their phosphorylation, a chemical-enzymatic approach for mapping the phosphoproteome, a perfectly specific inhibitor of any myosin motor protein, and developed isoform-selective inhibitors of phosphoinositol-lipid kinases.

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. The chemical-genetic approach for generation of monospecific inhibitors of any protein kinase is a powerful method for mapping cell signaling. In studies using these highly specific inhibitors (e.g., 1-NAPP1), we have found that signaling cascades are differentially dependent on the catalytic activity of kinases in a pathway. Specifically, partial inhibition of some kinases results in complete blockade of the entire cascade (e.g., cell cycle progression is highly sensitive to the catalytic activity of cyclin-dependent kinase 1), whereas equipotent inhibitors of other kinases in the same cascade are only capable of blocking a fraction of the output of a cascade (e.g., T cell signaling is minimally sensitive to blockade of Lck activity). Our working hypothesis is that the differential sensitivity of signaling cascades to inhibitors of different kinases is related to the quantitative relationship between signal output and catalytic activity of each kinase in a cascade. 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.

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 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. Identification of the ³²P-labeled proteins via traditional two-dimensional gel purification and mass spectrometry has identified hundreds of novel substrates of more than 50 widely divergent kinases, such as v-Src, CDK2, JNK, Cdc28, Erk2, Srb10, and Kin28. The remaining hurdle to the completion of the phosphoproteome map of all kinases is identification of the very low abundance (~100 molecules/cell) substrates. In the past year we have developed a new method for solving this problem.

The key to kinase-catalyzed delivery of an 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. We have developed a monoclonal antibody that is capable of uniquely recognizing the chemically derivatized phosphate analog on the surface of phosphoproteins. Although the electrophile reacts with other thiols, such as cysteines, only the adduct with the phosphorothiolate is specifically recognized by this monoclonal antibody. 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.

Last updated: September 15, 2008