biostudies-literatureTorosyan HUCSF Program for Breakthrough Biomedical ResearchNCI NIH HHSNIGMS NIH HHSU.S. Department of Health & Human Services | National Institutes of Health3543https://www.ebi.ac.uk/biostudies/studies/S-EPMC10279700biostudies-literatureUnknown14(1)PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, Nature communicationsStructural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3.PMC10279700T32 GM008284R35 GM139636U54 CA209891R35-GM139636Torosyan HVerba KAKrogan NJLo MJura NPaul MDDiwanji DForget APawlowski KfalseStructural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3.PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, 2023-01-01T00:00:00Z2023 Jun2026-05-29T07:59:04.029Z2024-10-18T02:16:24.143ZS-EPMC102797003733688310.1038/s41467-023-38864-0