Prediction and validation of new polarity substrates employing an aPKC anchoring motif
Authors: Constable Robert , Saiu Peter , Soriano Erika , Knowles Philip , Purkiss Andrew , O'Reilly Nicola , Riou Philippe , Parker Peter , McDonald Neil 
Published: November 2023

Objective

Atypical PKC is crucial to a variety of cell polarity processes and it's dysregulation through gene amplification has been implicated in Cancer1. Recent structural studies have revealed that in addition to a motif local to the phospho‐acceptor Ser/Thr, aPKC also recognises an FXR motif upstream of previously observed substrate contacts. Armed with knowledge of this motif we set out to predict new aPKC polarity substrates, which might use the motif for efficient recruitment, phosphorylation and proper cell polarity regulation. Here I report two substrates Prickle3 and Lulu1, which have been identified and validated as FXR type aPKCι substrates by peptide kinase assay, in cell phosphorylation and crystal structure.

Methods

In vitro kinase assays were performed using a Rhodamine labeled ParM ADP biosensor. Mammalian transient co‐transfections were performed in HEK 293 cells, phosphorylation was probed with an anti‐aPKC phosphospecific antibody and perturbed with the nucleotide competitive aPKC inhibitor.

Results

Predicted FXR type substrates found from sequence database searches were filtered for relevance to aPKC function by the criteria of membrane localization, phosphorylation site accessibility and implication in cell polarity. Twenty residue phosphorylation site peptides of Prickle3 and Lulu were shown to be phosphorylated by aPKCι in a kinase assay and mutational disruption of the FXR motif in these peptides to AXA was shown to reduce the catalytic efficiency of their phosphorylation. Transient transfection of HEK 293 cells with aPKCι and the substrate Lulu and Prickle3 indicated that aPKCι is capable of phosphorylating these substrates in cells and that this phosphorylation is sensitive to an aPKC selective inhibitor CRT0066854. A crystal structure of aPKCι in a ternary complex with Lulu1 residues 1–20 and Mg2+‐AMPPN indicates that the FXR motif of Lulu1 binds with the same geometry as that seen in the Par3‐Cr3 peptide, suggesting a role of the motif as a high affinity recruitment element for polarity substrates.

Conclusions

Knowledge of the FXR motif has enabled the prediction and validation of two novel aPKCι substrates and these experiments indicate that the motif enhances the efficiency of the phosphorylation of these substrates. Physiologically, the additional affinity conferred by the FXR motif to polarity substrates may play an important role in their recruitment to aPKC, efficient phosphorylation and subsequent regulation of cell polarity. Therefore future work will involve the dissection of the function of the motif in the context of the FXR substrate Par2 in C. elegans, by observation of the polarity phenotypes of motif disruptive mutant embryos.

Support or Funding Information

The Francis Crick Institute is a unique partnership between the Medical Research Council (MRC), Cancer Research UK, the Wellcome Trust, UCL (University College London), Imperial College London and King's College London.