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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.


Close-Up View of the Par3 CR3 Inhibitory Arm Pocket Bound to PKCιKD-2P with Other PKCιKD Structures(A) Close up of the inhibitory R+5 hook of Par3CR3 clamped by side chains Y265PKCι (P loop) and W298PKCι (αC helix) of PKCιKD-2P (gray cartoon, major interaction residues are shown as blue sticks). Key structural features of the PKCιKD-2P are labeled. Hydrogen bonds between key side chains are shown as dashed red lines.(B) Close up of R+5 pocket in the active conformation of AMPPCP-bound PKCιKD-2P structure. Hydrogen bonds between key side chains are shown as dashed red lines.(C) Close up of R+5 pocket in the previously solved ATP-bound PKCιKD-2P structure (PDB: 3A8W) (Takimura et al., 2010).(D) Close up of PKCιKD-1P K283R mutant within its ATP cleft (PDB: 4DC2) (Wang et al., 2012a).See also Figure S3C for refined nucleotide analog electron density and Figure S3D for a comparison with a chemical inhibitor-induced PKCιKD-2P conformer.
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fig3: Close-Up View of the Par3 CR3 Inhibitory Arm Pocket Bound to PKCιKD-2P with Other PKCιKD Structures(A) Close up of the inhibitory R+5 hook of Par3CR3 clamped by side chains Y265PKCι (P loop) and W298PKCι (αC helix) of PKCιKD-2P (gray cartoon, major interaction residues are shown as blue sticks). Key structural features of the PKCιKD-2P are labeled. Hydrogen bonds between key side chains are shown as dashed red lines.(B) Close up of R+5 pocket in the active conformation of AMPPCP-bound PKCιKD-2P structure. Hydrogen bonds between key side chains are shown as dashed red lines.(C) Close up of R+5 pocket in the previously solved ATP-bound PKCιKD-2P structure (PDB: 3A8W) (Takimura et al., 2010).(D) Close up of PKCιKD-1P K283R mutant within its ATP cleft (PDB: 4DC2) (Wang et al., 2012a).See also Figure S3C for refined nucleotide analog electron density and Figure S3D for a comparison with a chemical inhibitor-induced PKCιKD-2P conformer.

Mentions: Site 3 contains carboxy-terminal flanking residues to the P site stretching from S+2 to T+6. We define this portion of Par3CR3 as the “inhibitory arm,” as it directly perturbs an active PKCιKD-2P N-lobe conformation (discussed later). Residue K+4 directly contacts pT412PKCι of the activation loop enhancing the recognition of mature, primed PKC ιKD-2P, but importantly not a partially primed PKCιKD-1P. Crucially, the R+5 side chain is buried within a hydrophobic pocket beneath the regulatory αC helix. The pocket is lined by side chains from Y265PKCι on the glycine loop and W298PKCι of the αC helix, each making π-stacking interactions with the guanidino group of R+5 (Figures 2C and 3A). Both aromatic side chains are unique to aPKC isozymes from Drosophila to mammals. Finally, T+6 (equivalent to T833Par3, a known ROCK-driven phosphorylation site discussed later) lies adjacent to an acidic patch within the αB helix making side-chain and main-chain contacts to D295PKCι and a Mg ion (Figures 2C and 3A). Overall, the structure reveals that the Par3CR3 clamp involves an “inhibitory arm” and an “anchoring arm,” which together recognize and inhibit a nucleotide-bound PKCιKD-2P conformer.


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Close-Up View of the Par3 CR3 Inhibitory Arm Pocket Bound to PKCιKD-2P with Other PKCιKD Structures(A) Close up of the inhibitory R+5 hook of Par3CR3 clamped by side chains Y265PKCι (P loop) and W298PKCι (αC helix) of PKCιKD-2P (gray cartoon, major interaction residues are shown as blue sticks). Key structural features of the PKCιKD-2P are labeled. Hydrogen bonds between key side chains are shown as dashed red lines.(B) Close up of R+5 pocket in the active conformation of AMPPCP-bound PKCιKD-2P structure. Hydrogen bonds between key side chains are shown as dashed red lines.(C) Close up of R+5 pocket in the previously solved ATP-bound PKCιKD-2P structure (PDB: 3A8W) (Takimura et al., 2010).(D) Close up of PKCιKD-1P K283R mutant within its ATP cleft (PDB: 4DC2) (Wang et al., 2012a).See also Figure S3C for refined nucleotide analog electron density and Figure S3D for a comparison with a chemical inhibitor-induced PKCιKD-2P conformer.
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Related In: Results  -  Collection

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fig3: Close-Up View of the Par3 CR3 Inhibitory Arm Pocket Bound to PKCιKD-2P with Other PKCιKD Structures(A) Close up of the inhibitory R+5 hook of Par3CR3 clamped by side chains Y265PKCι (P loop) and W298PKCι (αC helix) of PKCιKD-2P (gray cartoon, major interaction residues are shown as blue sticks). Key structural features of the PKCιKD-2P are labeled. Hydrogen bonds between key side chains are shown as dashed red lines.(B) Close up of R+5 pocket in the active conformation of AMPPCP-bound PKCιKD-2P structure. Hydrogen bonds between key side chains are shown as dashed red lines.(C) Close up of R+5 pocket in the previously solved ATP-bound PKCιKD-2P structure (PDB: 3A8W) (Takimura et al., 2010).(D) Close up of PKCιKD-1P K283R mutant within its ATP cleft (PDB: 4DC2) (Wang et al., 2012a).See also Figure S3C for refined nucleotide analog electron density and Figure S3D for a comparison with a chemical inhibitor-induced PKCιKD-2P conformer.
Mentions: Site 3 contains carboxy-terminal flanking residues to the P site stretching from S+2 to T+6. We define this portion of Par3CR3 as the “inhibitory arm,” as it directly perturbs an active PKCιKD-2P N-lobe conformation (discussed later). Residue K+4 directly contacts pT412PKCι of the activation loop enhancing the recognition of mature, primed PKC ιKD-2P, but importantly not a partially primed PKCιKD-1P. Crucially, the R+5 side chain is buried within a hydrophobic pocket beneath the regulatory αC helix. The pocket is lined by side chains from Y265PKCι on the glycine loop and W298PKCι of the αC helix, each making π-stacking interactions with the guanidino group of R+5 (Figures 2C and 3A). Both aromatic side chains are unique to aPKC isozymes from Drosophila to mammals. Finally, T+6 (equivalent to T833Par3, a known ROCK-driven phosphorylation site discussed later) lies adjacent to an acidic patch within the αB helix making side-chain and main-chain contacts to D295PKCι and a Mg ion (Figures 2C and 3A). Overall, the structure reveals that the Par3CR3 clamp involves an “inhibitory arm” and an “anchoring arm,” which together recognize and inhibit a nucleotide-bound PKCιKD-2P conformer.

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.