Similar to what has been observed for PEAK2, the function of PEAK3 is cell type-dependent. In fibroblast cells, PEAK3 was found to directly interact with CrkII and inhibit CrkII-dependent membrane ruffling 15, 16. PEAK3, the newest member of the PEAK pseudokinase family, and most distinct in its primary sequence, remained unannotated as a kinase until recently, and its functional characterization lags behind PEAK1 and PEAK2 15, 16. The emerging understanding of PEAK family signaling is that PEAKs regulate distinct cellular outcomes by selectively engaging their interaction partners. PEAK2/Csk binding increases Csk localization and activity at focal adhesions, inducing cell elongation and motility 13, 14. However, in epithelial cells, PEAK2 activity induces cell growth upon binding the C-terminal Src kinase (Csk), as a result of Src-dependent phosphorylation 9, 10. PEAK2 was initially found to increase RhoA signaling by interacting with the Rho family GTPase Rnd2, resulting in the inhibition of neurite outgrowth 12. PEAK1 binding to Shc1 results in a switch in the Shc1-associated proteome from factors involved in pro-mitogenic and pro-survival signaling to proteins involved in cytoskeletal reorganization, migration, and eventually signal termination 11. Notably, PEAK1 has been shown to be important for the modulation of the late phase of EGF-induced EGFR signaling via its interaction with Shc1 11. PEAK1 overexpression increases cell motility and migration, likely due to a combination of a PEAK1-dependent increase in ERK signaling and through an ERK-independent mechanism via direct association of PEAK1 with the Src/p130Cas/Crk complex 9, 10. The three PEAKs act as molecular scaffolds in pathways involved primarily in cell motility and migration 7, 8. The pseudopodium-enriched atypical kinase (PEAK) family of pseudokinases, also known as New Kinase Family 3 (NKF3), is composed of three members: PEAK1 (also known as Sugen kinase 269), PEAK2 (also known as Pragmin or SgK223), and PEAK3 (also known as chromosome 19 Open Reading Frame 35 ). Although pseudokinases are increasingly recognized as essential regulators of cellular signal transduction, the molecular mechanisms of most of their non-canonical kinase functions remain poorly understood. Instead, these catalytically inactive kinases have evolved other functionalities to propagate signaling, including allosteric modulation and scaffolding of their interaction partners 5, 6. However, a significant subset of human kinases, termed pseudokinases, lack this vital enzymatic function due to the presence of inactivating mutations within key catalytic motifs.

Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity.īy catalyzing phosphorylation, protein kinases play important roles in regulating essential cellular processes, such as cell survival, proliferation, migration, and organogenesis 1, 2, 3, 4.

Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. 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. 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. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression.

Scaffold protein h1