A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations.

The PEAK protein family, comprising PEAK1-3, are pseudokinase scaffolds that regulate cell proliferation and motility via recruitment of specific effectors. For PEAK3, the latter include the adaptor proteins Grb2 and CrkII and the Arf GTPase-activating protein (ArfGAP) ASAP1. PEAK3 exhibits a tandem site spanning a CrkII SH3 domain binding sequence and phosphorylation-dependent 14-3-3 recruitment motif at serine 69 (S69), with 14-3-3 binding mediating a negative control 'switch' on PEAK3 signaling. However, whether this control switch is subject to (patho)physiological regulation has remained unclear. Here, using MCF-10A breast epithelial cells as a model system, we demonstrate that S69 phosphorylation occurs predominantly in the cytoplasm and is subject to growth factor regulation, being enhanced by EGF and insulin stimulation but with distinct temporal dynamics. We identify Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) as key mediators of this phosphorylation event. Disruption of the pS69/14-3-3 interaction in the PEAK3 S69A mutant leads to elevated basal Erk phosphorylation, altered EGF-induced Erk and Akt activation kinetics, partial epithelial-to-mesenchymal transition, and increased Arf1 activation. Interrogation of the COSMIC database identified cancer-associated mutations in the tandem recruitment site, and functional characterization revealed a subset that confer enhanced Grb2/ASAP1 binding and migration potential compared with wild-type PEAK3. Interestingly, amongst the cancer mutations, PEAK3 R66P and R66Q lost 14-3-3 binding in vivo, but L55P retained it. Together, our study reveals that the tandem PEAK3 regulatory site is subject to physiological control and is also mutated in cancer.