Dataset Information

The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.

ABSTRACT: The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding motifs are involved in these interactions. This is a prerequisite to finely analyze how these pathways are integrated in the cell machinery and to study their role in cancer and other human diseases when this network of interactions is perturbed. In this work, the domain and the binding motif of fourteen proteins interacting with SKAP2 were precisely defined and a new interactor, FAM102A was discovered. Herein, a fine-tuning between the binding of SRC kinases and their activation was identified. This last process, which depends on SKAP2 dimerization, indirectly affects the binding of FYB protein. Analysis of conformational changes associated with activation/inhibition of SRC family members, presently limited to their effect on kinase activity, is extended to their interactive network, which paves the way for therapeutic development.

SUBMITTER: Bureau JF

PROVIDER: S-EPMC5862564 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.

Bureau Jean-François JF Cassonnet Patricia P Grange Laura L Dessapt Julien J Jones Louis L Demeret Caroline C Sakuntabhai Anavaj A Jacob Yves Y

Oncotarget 20180206 17

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding mo ...[more]

PMID: 29568343

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Project description:The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct "open" and "closed" conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

Dataset Information

The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.

Publications

The SRC-family tyrosine kinase HCK shapes the landscape of SKAP2 interactome.

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