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 Mg 2+ ‐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.

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.

AMP-activated protein kinase (AMPK) has an important role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels, it activates energy-producing pathways and inhibits energy-consuming processes. Here, a role for ADP in regulating AMPK by protecting the enzyme from dephosphorylation is defined, and a crystal structure of the active enzyme containing the kinase domain is presented. A model is proposed for how AMP and ADP regulate AMPK activity. The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer2,3,4,5,6. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain7: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation, as well as causing allosteric activation9. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.

MP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. This crystal structure of the trimeric regulatory fragment of mammalian AMPK reveals the modes of AMP and ATP binding.