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aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization

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ABSTRACT

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.

No MeSH data available.


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Proposed Model for Par3/Baz Antagonism and PolarizationWe propose two states for PKC-Par3/Baz interaction driven by aPKC kinase domain and Par3/Baz CR3 region. A high-affinity interaction is inhibitory and requires both arms flanking the PKC consensus motif of the CR3 region. By engaging pockets within both the N and C lobes of aPKC kinase domain, Par3/Baz phosphorylation is prevented but Par6-aPKC is recruited to the apical membrane. In an activated state, aPKC is resistant to CR3 antagonism due either to an inaccessible αC helix pocket or a CR3 interaction being destabilized by phosphorylation of T833Par3 or by aPKC lacking a PDK1-driven T412PKCι phosphorylation. Either of these possibilities could result in Par3/Baz binding as a substrate exposing its PKC consensus site R-X-S-Ψ to phospho-transfer. Phosphorylated Par3/Baz is then excluded from the Par complex and thus from the apical membrane domain, and relocalizes to AJs.
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fig7: Proposed Model for Par3/Baz Antagonism and PolarizationWe propose two states for PKC-Par3/Baz interaction driven by aPKC kinase domain and Par3/Baz CR3 region. A high-affinity interaction is inhibitory and requires both arms flanking the PKC consensus motif of the CR3 region. By engaging pockets within both the N and C lobes of aPKC kinase domain, Par3/Baz phosphorylation is prevented but Par6-aPKC is recruited to the apical membrane. In an activated state, aPKC is resistant to CR3 antagonism due either to an inaccessible αC helix pocket or a CR3 interaction being destabilized by phosphorylation of T833Par3 or by aPKC lacking a PDK1-driven T412PKCι phosphorylation. Either of these possibilities could result in Par3/Baz binding as a substrate exposing its PKC consensus site R-X-S-Ψ to phospho-transfer. Phosphorylated Par3/Baz is then excluded from the Par complex and thus from the apical membrane domain, and relocalizes to AJs.

Mentions: Our results reveal the molecular basis for Par3 antagonism of aPKC through high-affinity inhibitory CR3 arm interactions that span both N and C lobes of the PKCι kinase domain. Our structural and biochemical data provide a model that supports a mechanism explaining apical-junctional polarization of Par3/Baz in epithelial cells (Figure 7). Previous work has shown that apical localization of Par3/Baz depends on it being part of the Par complex, where Par3/Baz is not phosphorylated by aPKC, while junctional localization of Par3/Baz occurs when it is phosphorylated by aPKC (Morais-de-Sa et al., 2010, Walther and Pichaud, 2010). Formation of the Par complex with Par3/Baz is known to be crucial for apical membrane recruitment of aPKC-Par6 (Gao et al., 2002).


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Proposed Model for Par3/Baz Antagonism and PolarizationWe propose two states for PKC-Par3/Baz interaction driven by aPKC kinase domain and Par3/Baz CR3 region. A high-affinity interaction is inhibitory and requires both arms flanking the PKC consensus motif of the CR3 region. By engaging pockets within both the N and C lobes of aPKC kinase domain, Par3/Baz phosphorylation is prevented but Par6-aPKC is recruited to the apical membrane. In an activated state, aPKC is resistant to CR3 antagonism due either to an inaccessible αC helix pocket or a CR3 interaction being destabilized by phosphorylation of T833Par3 or by aPKC lacking a PDK1-driven T412PKCι phosphorylation. Either of these possibilities could result in Par3/Baz binding as a substrate exposing its PKC consensus site R-X-S-Ψ to phospho-transfer. Phosphorylated Par3/Baz is then excluded from the Par complex and thus from the apical membrane domain, and relocalizes to AJs.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4998004&req=5

fig7: Proposed Model for Par3/Baz Antagonism and PolarizationWe propose two states for PKC-Par3/Baz interaction driven by aPKC kinase domain and Par3/Baz CR3 region. A high-affinity interaction is inhibitory and requires both arms flanking the PKC consensus motif of the CR3 region. By engaging pockets within both the N and C lobes of aPKC kinase domain, Par3/Baz phosphorylation is prevented but Par6-aPKC is recruited to the apical membrane. In an activated state, aPKC is resistant to CR3 antagonism due either to an inaccessible αC helix pocket or a CR3 interaction being destabilized by phosphorylation of T833Par3 or by aPKC lacking a PDK1-driven T412PKCι phosphorylation. Either of these possibilities could result in Par3/Baz binding as a substrate exposing its PKC consensus site R-X-S-Ψ to phospho-transfer. Phosphorylated Par3/Baz is then excluded from the Par complex and thus from the apical membrane domain, and relocalizes to AJs.
Mentions: Our results reveal the molecular basis for Par3 antagonism of aPKC through high-affinity inhibitory CR3 arm interactions that span both N and C lobes of the PKCι kinase domain. Our structural and biochemical data provide a model that supports a mechanism explaining apical-junctional polarization of Par3/Baz in epithelial cells (Figure 7). Previous work has shown that apical localization of Par3/Baz depends on it being part of the Par complex, where Par3/Baz is not phosphorylated by aPKC, while junctional localization of Par3/Baz occurs when it is phosphorylated by aPKC (Morais-de-Sa et al., 2010, Walther and Pichaud, 2010). Formation of the Par complex with Par3/Baz is known to be crucial for apical membrane recruitment of aPKC-Par6 (Gao et al., 2002).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus