Structure-function studies of protein tyrosine kinases: Regulation and substrate specificity

Protein tyrosine kinases (PTKs) are a large family of enzymes that play critical roles in signal transduction. Through transferring γ-phosphate group from ATP to protein substrates, PTKs regulate functions of these substrates. Due to the utmost importance in signal transduction, malfunction of PTKs usually leads to severe consequences such as cell transformation. Therefore, how PTKs function becomes a focal question in the field of cancer biology. To build a knowledge base for the investigation of specific functions of PTKs, we focused on two major aspects: regulation and substrate specificity. Two general strategies of regulating PTKs have been observed. In the first strategy, the PTK catalytic domain such as Src is intrinsically active, which can be suppressed by regulatory domains. In the second strategy, the catalytic domain such as Csk is intrinsically inactive and can be activated by regulatory domains. In this study, we identified an activating switch that keeps Src catalytic domain active and that is missing in Csk. This switch consists of three essential structural components in the ATP-binding lobe: Arg264 residue, a cluster of residues in α-helix C region, and β-4.5 turn region. Csk catalytic domain was activated by grafting these three components. These results reveal an important structure that dictates the regulatory strategy of a kinase. Each PTK phosphorylates a unique spectrum of substrates. Disruption of recognition between PTKs and their substrates is generally believed as a promising strategy of blocking kinase function and a potential therapeutic strategy of curing certain types of cancer. In this study, we characterized how Abl kinase recognizes its physiological substrate Crk L. One of the proline-rich motifs on Abl carboxyl-terminal region interacts with Crk L amino-terminal SH3 domain, recruiting Crk L as substrate and also making the phosphorylation site more accessible. This interaction is assisted by Crk L SH2 domain in a nonclassical manner. Simultaneously, Abl directly recognizes specific sequence of the phosphorylation sites, facilitating the recruitment of Crk L. This comprehensive analysis of Abl-Crk L interaction expands current understanding of PTK-substrate recognition, substantiating the molecular foundation of PTK inhibitor design