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The Src family kinases (SFKs) are an important family of tyrosine kinases that are allosterically regulated by their SH2 and SH3 domains. Engagement of SFK SH2 and SH3 domains with their intramolecular ligands leads to reduced kinase activity by stabilizing an inactive ATP-binding site conformation. Disruption of these intramolecular interactions stabilizes a more active ATP-binding site conformation and restores SFK activity. Interestingly, this allosteric relationship is bidirectional in that ATP-competitive ligands that stabilize distinct active site conformations can divergently modulate the abilities of the regulatory SH2 and SH3 domains to participate in intermolecular interactions. Here, we describe a series of assays that profile the bidirectional relationship between the ATP-binding sites and regulatory domains of SFKs. These methods can be used to discover ATP-competitive inhibitors that are selective for distinct ATP-binding site conformations of SFKs and for characterizing the effects that ATP-competitive inhibitors of SFKs have on domains that are distal to their site of interaction.

Identifying cellular targets of bioactive small molecules from large-scale screening campaigns can be a significant bottleneck in developing novel therapeutics. Our rapid small-molecule target profiling protocol combines affinity enrichment and SILAC for proteomic identification of small molecule-protein interactions. Selective interactions are easily discernable from nonspecific protein binding by quantitative ratios. Using kinase inhibitors as an example, we provide an optimized protocol featuring on-bead protein digestion and single nano-flow liquid chromatographic-mass spectrometric (LC-MS) analyses, consequently increasing analytical throughput and sensitivity over gel-based sample preparation methods for rapid profiling of kinase inhibitor targets.

There is an allosteric relationship between the kinase and RNase domains of the ER stress sensor IRE1α. This relationship has been exploited to develop ATP-competitive inhibitors that are able to divergently modulate the RNase activity of IRE1α through its kinase domain. Here, we describe a series of biochemical methods for profiling the dual enzymatic activities of IRE1α. These methods can be used to ascertain how ATP-competitive inhibitors affect the kinase activity of IRE1α and for determining whether these ligands allosterically activate or inactivate RNase activity.

Recent interest in inactive kinase conformations has generated the need to develop new biochemical tools to study them. Here, we describe the use of a fluorescent probe that selectively and potently binds to a specific inactive conformation of protein kinases. This allows for the thermodynamics and kinetics of ligand binding to be determined.

Several protein kinases have been characterized in a specific inactive form called the DFG-out conformation. Unlike the active conformation which is conserved in all kinases, the inactive DFG-out conformation appears to be accessible to only certain kinases. This inactive conformation has been successfully targeted with highly selective kinase inhibitors, including the cancer drugs imatinib and sorafenib. However, the structural and sequence requirements for adopting this conformation are still poorly understood. Here, we describe a general method for enriching DFG-out adopting kinases from cell lysates with an affinity resin that contains a general ligand that specifically recognizes this inactive form.