biostudies-literatureRoy MJDepartment of Education and Training | Australian Research CouncilDepartment of Health | National Health and Medical Research Council3542https://www.ebi.ac.uk/biostudies/studies/S-EPMC10279719biostudies-literatureUnknown14(1)PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide aNature communicationsStructural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling.PMC102797192008096DP220103638IRIISS (9000719)DP190103672APP1144149Roy MJKropp ASurudoi MGHardy JMLucet ISDai WMa XCotton TRHou JDite TAPatel OLechtenberg BCDaly RJLiang LYfalseStructural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling.PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a2023-01-01T00:00:00Z2023 Jun2026-05-28T20:30:57.816Z2024-11-07T10:02:34.805ZS-EPMC102797193733688410.1038/s41467-023-38869-9