Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates an ‘actionable’ signalling axis in triple negative breast cancer

To characterize the interactomes of PEAK1 and PEAK2 homodimers, and the PEAK1/PEAK2 heterodimer, we applied bimolecular complementation affinity purification coupled with tandem mass spectrometry (BiCAP-MS/MS), a recently-developed technique that specifically identifies the interactors of any pair of interacting proteins while excluding those binding to individual components. Appropriate pairs of V1- and V2-tagged proteins were co-transfected into HEK293T cells, and then specific dimeric complexes were affinity purified with GFP-Trap nanobody and associated proteins identified by LC-MS/MS. Proteins exhibiting significantly increased abundance in particular PEAK dimers versus the Venus control were identified by label-free quantitative MS analysis, and are presented in volcano plots.