Abstract
SummaryAtypical 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.
Highlights
Epithelial tissues are composed of sheets of polarized cells that are connected by adherens junctions (AJs) (Laprise and Tepass, 2011; St Johnston and Ahringer, 2010; Suzuki and Ohno, 2006)
The Par3 CR3 Region Inhibits Nucleotide-Bound Primed PKCi Kinase Domain through Two Flanking Arm Contacts The human Par3 conserved region 3 (CR3, covering residues 816–834, defined hereafter as Par3CR3) is able to bind to PKCi (Nagai-Tamai et al, 2002) and contains a phospho-acceptor site (P site) at residue serine 827 known to be phosphorylated by PKCi (Figures 1A and 1B)
We found that Par3CR3 strongly inhibited the catalytic activity of PKC-iota kinase domain (PKCiKD)-2P in vitro and could competitively block phosphorylation of a model substrate peptide, with an apparent 50% inhibitory concentration (IC50) of 0.45 ± 0.18 mM
Summary
Epithelial tissues are composed of sheets of polarized cells that are connected by adherens junctions (AJs) (Laprise and Tepass, 2011; St Johnston and Ahringer, 2010; Suzuki and Ohno, 2006). The role of Par3/Baz at AJs is thought to be essential as it involves defining the position of AJs during the establishment of epithelial polarity (Wang et al, 2012b) and possibly remodeling of AJs as tissues undergo morphogenetic change (Walther and Pichaud, 2010) The regulation of this switch of Par3/Baz subcellular localization from the apical membrane to AJs has been shown in Drosophila to be dependent on aPKC phosphorylating Par3/Baz on serine 980 in vivo (Morais-de-Sa et al, 2010; Walther and Pichaud, 2010). A similar conundrum exists whereby Par is critical for the recruitment of PKCi to the apical membrane and is known to be an in vivo substrate of PKCi, but loss of Par in transformed epithelial cells can lead to PKCi activation
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