A kinetic approach to distinguish between catalytic mechanisms of protein kinase autophosphorylation

Abstract

Protein kinase autoactivation, via phosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation‐dependent signaling cascades. Their molecular mechanisms can be simply categorized to intra‐molecular(in cis) or inter‐molecular(in trans). Specifically, the trans‐autophosphorylation kinases vary in their initiation modes. In this study, we give a full analysis for the four initiation modes in trans‐autophosphorylation and develop a kinetic approach, which is based on the kinetic theory of substrate reaction during modification of enzyme activity, to quantitively distinguish them. Applying our kinetic method to p21‐activated kinase 2 kinase domain(PAK2‐KD), we identified the molecular mechanism of PAK2‐KD trans‐autophosphorylation and determined all microscopic kinetic constants for its autoactivation process, coincident with our structural studies on PAK1 kinase domain. The unphosphorylated PAK2‐KD, which is lack of structural constraints in context, could dynamically adopt both the active and inactive conformations, thus initiates the autoactivation process differently from the full‐length PAK2. Our kinetic method gives a general solution to describe distinct catalytic mechanisms of protein kinase autophosphorylation and is particular useful during the fast modification reaction.