Distinct PEAK3 interactors and outputs expand the signaling potential of the PEAK pseudokinase family

The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr 24 , likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal-diversifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.