PDK1:PKCa heterodimer association-dissociation dynamics in single-molecule diffusion tracks on a target membrane

Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCa on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCa het-erodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCa molecules bind and unbind. In the absence of PKCa, a membrane-associated PDK1 molecule exhibits high diffu-sivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP3 lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCa contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCa heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCa heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCa heterodimer has been shown to competitively inhibit PDK1 phos-phoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCa association-dissociation kinetics has important bio-logical and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.

Automated summary

This study investigates the association-dissociation kinetics of the PDK1:PKCa heterodimer. The findings reveal that single PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between the PDK1 monomer and the PDK1:PKCa heterodimer. The elucidation of these kinetics has important biological and medical implications.