Structural insightsStructural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3 into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

ABSTRACT: The three members of the PEAK family of pseudokinases (PEAK1, PEAK2, and PEAK3) are molecular scaffolds that have recently emerged as important nodes in signaling pathways that control cell migration, morphology, and proliferation, and are increasingly found mis-regulated in human cancers. While no structures of PEAK3 have been solved to date, crystal structures of the PEAK1 and PEAK2 pseudokinase domains revealed their dimeric organization. It remains unclear how dimerization plays a role in PEAK scaffolding functions as no structures of PEAK family members in complex with their binding partners have been solved. Here, we report the cryo-EM structure of the PEAK3 pseudokinase, also adopting a dimeric state, and in complex with an endogenous 14-3-3 heterodimer purified from mammalian cells. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the Split HElical Dimerization (SHED) domain of the PEAK3 dimer. The binding interface is comprised of a canonical primary interaction involving two phosphorylated 14-3-3 consensus binding sites located in the N-terminal domains of the PEAK3 monomers docked in the conserved amphipathic grooves of the 14-3-3 dimer, and a unique secondary interaction between 14-3-3 and PEAK3 that has not been observed in any previous structures of 14-3-3/client complexes. Disruption of these interactions results in the relocation of PEAK3 to the nucleus and changes its cellular interactome. Lastly, we identify Protein Kinase D as the regulator of PEAK3/14-3-3 binding, providing a mechanism by which the diverse functions of the PEAK3 scaffold might be fine-tuned in cells.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Antoine Forget  

LAB HEAD: Nevan Krogan

PROVIDER: PXD035574 | Pride | 2023-07-20

REPOSITORIES: Pride