Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase with a complex activation mechanism.
• Autoinhibition by the FERM domain is a key regulatory feature, but the dynamics of activation remain incompletely understood.

Purpose of the Study:

• To elucidate the conformational dynamics of Pyk2 during activation, focusing on autophosphorylation and Src kinase-mediated phosphorylation.
• To map dynamic changes associated with substrate binding and activation loop phosphorylation.

Main Methods:

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to probe protein dynamics.
• Kinase activity profiling and targeted mutagenesis were employed to assess functional consequences.

Main Results:

• Nucleotide binding stabilizes the autoinhibitory state, while phosphorylation by Src kinase deprotects regulatory surfaces.
• Phosphorylation induces conformational rearrangements, organizing active site motifs and stabilizing the active conformation by preventing FERM domain re-interaction.
• Mutagenesis confirmed that phosphorylation-driven rearrangements are crucial for elevating kinase activity.

Conclusions:

• Pyk2 activation is a multistage process involving dynamic conformational shifts regulated by phosphorylation.
• Understanding these dynamics provides insights into tyrosine kinase regulation and potential therapeutic strategies.