Single-molecule studies reveal regulatory interactions between master kinases PDK1, AKT1, and PKC

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP₃ also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP₃ and Ca²⁺ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP₃, whereas PKCα is recruited to the membrane by Ca²⁺. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP₃-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.