Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling

Abstract

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 a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

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

“…Here, like Grb2, they were identified as interactors of the PEAK1 homodimer and PEAK1/PEAK2 heterodimer, with another PP1 family member, PPP1CB, associating with the PEAK1 homodimer. Of interest given a recent paper reporting a key regulatory role for 14-3-3 proteins in regulating PEAK3 effector recruitment and biological activity 24 , multiple 14-3-3 isoforms bound both homodimers and the heterodimer (Supp. Table 1).…”

“…We utilized this approach since it more selectively addresses the impact of CAMK2-mediated PEAK1 phosphorylation, while RA306 treatment would exert a more general inhibition of CAMK2-mediated cellular phosphorylation. Binding of the adaptors Crk-L and Grb2, which associate with PEAK1 via SH3 and SH2 domain-mediated interactions, respectively 9,24 , was unaffected by the CIM mutations (Supp. Fig.…”

“…This is consistent with the overlap between the minimal CAMK2 phosphorylation site consensus (R/KXXS/T) and the 14-3-3 mode 1 motif for binding, RXXpS/TXP. In the case of PEAK3, 14-3-3 binding to phosphorylated PEAK3 S69 impairs Grb2 and Crk recruitment to the scaffold, and the PEAK3 S69A mutant exhibits markedly enhanced ability to promote cell migration 24 . With PEAK1, the overall effect of CAMK2-mediated phosphorylation on PEAK1-promoted migration/invasion is positive, so 14-3-3 binding may downregulate binding of negative regulatory factors, and/or increase association with positive ones, such as PEAK2.…”

Location, location, location: Protein kinase nanoclustering for optimised signalling output

“…The pseudokinases PEAK1/3 are important scaffolding regulators for the EGFR signalling ( Paul et al, 2022 ). A recent study investigating the PEAK3 interactome revealed that it acts as a dynamic scaffold together with essential adaptor proteins to regulate signal transduction ( Roy et al, 2023 ). Lipid raft environment has been shown to enhance LYN kinase activity ( Young et al, 2003 ).…”

The Configuration of GRB2 in Protein Interaction and Signal Transduction

“…This structure includes a central SH2 domain flanked by two Src homologous 3 (SH3) domains on both sides [ 8 ]. The SH2 domain, spanning amino acids 60 to 152, is composed of 93 amino acids and enables GRB2 to recognize the phosphotyrosine consensus motif pYxNx (where Y is tyrosine, x represents any natural amino acid, and N is asparagine) in activated RTKs and other receptors [ 9 ]. On the other hand, there are two SH3 domains in GRB2, including a carboxyl-terminal SH3 (c-SH3) domain (residues 1–58) and an amino-terminal SH3 (n-SH3) domain (residues 156–215), both of which can interact with proline-rich motifs such as PxxP motifs (where P is proline), considered as a linker between the SH2 domain and downstream protein ( Figure 1 b,c) [ 10 ].…”