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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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Structural Basis for Par3/Baz CR3-Mediated Inhibition of aPKC(A) Overall structure of PKCιKD-2P (gray surface) bound to Par3CR3 (green stick) with Mg-AMP-PNP shown as an orange surface and priming sites at T564 and T412 indicated.(B) Schematic representation of three sites of contacts between Par3CR3 and PKCιKD-2P. The known phospho-acceptor site at serine 0 is shown in red.(C) Close up of the contacts between Par3CR3 peptide (green) and PKCιKD-2P (residues making contacts shown in blue). Hydrogen bonds between side chains or main-chain atoms are shown as dashed red lines. Pink spheres represent magnesium ions.See also Figures S3A and S3B for refined Par3CR3 electron density and intramolecular hydrogen bonds within Par3CR3.
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fig2: Structural Basis for Par3/Baz CR3-Mediated Inhibition of aPKC(A) Overall structure of PKCιKD-2P (gray surface) bound to Par3CR3 (green stick) with Mg-AMP-PNP shown as an orange surface and priming sites at T564 and T412 indicated.(B) Schematic representation of three sites of contacts between Par3CR3 and PKCιKD-2P. The known phospho-acceptor site at serine 0 is shown in red.(C) Close up of the contacts between Par3CR3 peptide (green) and PKCιKD-2P (residues making contacts shown in blue). Hydrogen bonds between side chains or main-chain atoms are shown as dashed red lines. Pink spheres represent magnesium ions.See also Figures S3A and S3B for refined Par3CR3 electron density and intramolecular hydrogen bonds within Par3CR3.

Mentions: To understand how Par3CR3 could inhibit PKCιKD-2P, we determined the 2.0-Å crystal structure of a longer Par3 peptide (residues 816–841) bound to PKCιKD-2P and Mg-AMPPNP (adenylyl imidodiphosphate) (Figures 2A and S3A; Table 1). The Par3CR3 peptide is well ordered in this structure and contains seven intramolecular hydrogen bonds (Figure S3B). It engages PKCιKD-2P by adopting a “staple”-shaped conformation with two arms that flank the S827Par3 phospho-acceptor site. Each arm binds in close proximity to opposite ends of the nucleotide, suggesting that recognition of aPKC is driven by nucleotide occupancy. The relative orientation of N and C lobes indicates a “closed” rather than “open” conformation. Par3CR3 contacts extend from a pocket beneath the ribose-binding pocket of PKCι (site 1), across the G helix (site 2) through to the activation loop, αB and αC helices of the PKCιKD-2P N lobe (site 3) (Figures 2A and 2B). A total surface area of more than 1,305 Å2 is buried within the complex, consistent with a high-affinity inhibitory interaction. The nucleotide cleft is occupied by an Mg-AMPPNP nucleotide (Figure 2C). The conserved nucleotide-coordinating lysine (K283PKCι) forms a salt bridge with the conserved αC-helix glutamate (E302PKCι) side chain found in many active kinase conformers (Kornev et al., 2008). The terminal γ-phosphate of AMPPNP is not observed in the structure, consistent with AMPPNP being rapidly hydrolyzed under the crystallization conditions (see Experimental Procedures). A magnesium ion, equivalent to Mg2 of PKA, is present, bridging both the α and β phosphates of AMPPNP (Adams and Taylor, 1993, Zheng et al., 1993). The Mg1 ion is not present, as frequently found in ADP-complexed AGC kinase structures.


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Structural Basis for Par3/Baz CR3-Mediated Inhibition of aPKC(A) Overall structure of PKCιKD-2P (gray surface) bound to Par3CR3 (green stick) with Mg-AMP-PNP shown as an orange surface and priming sites at T564 and T412 indicated.(B) Schematic representation of three sites of contacts between Par3CR3 and PKCιKD-2P. The known phospho-acceptor site at serine 0 is shown in red.(C) Close up of the contacts between Par3CR3 peptide (green) and PKCιKD-2P (residues making contacts shown in blue). Hydrogen bonds between side chains or main-chain atoms are shown as dashed red lines. Pink spheres represent magnesium ions.See also Figures S3A and S3B for refined Par3CR3 electron density and intramolecular hydrogen bonds within Par3CR3.
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Related In: Results  -  Collection

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fig2: Structural Basis for Par3/Baz CR3-Mediated Inhibition of aPKC(A) Overall structure of PKCιKD-2P (gray surface) bound to Par3CR3 (green stick) with Mg-AMP-PNP shown as an orange surface and priming sites at T564 and T412 indicated.(B) Schematic representation of three sites of contacts between Par3CR3 and PKCιKD-2P. The known phospho-acceptor site at serine 0 is shown in red.(C) Close up of the contacts between Par3CR3 peptide (green) and PKCιKD-2P (residues making contacts shown in blue). Hydrogen bonds between side chains or main-chain atoms are shown as dashed red lines. Pink spheres represent magnesium ions.See also Figures S3A and S3B for refined Par3CR3 electron density and intramolecular hydrogen bonds within Par3CR3.
Mentions: To understand how Par3CR3 could inhibit PKCιKD-2P, we determined the 2.0-Å crystal structure of a longer Par3 peptide (residues 816–841) bound to PKCιKD-2P and Mg-AMPPNP (adenylyl imidodiphosphate) (Figures 2A and S3A; Table 1). The Par3CR3 peptide is well ordered in this structure and contains seven intramolecular hydrogen bonds (Figure S3B). It engages PKCιKD-2P by adopting a “staple”-shaped conformation with two arms that flank the S827Par3 phospho-acceptor site. Each arm binds in close proximity to opposite ends of the nucleotide, suggesting that recognition of aPKC is driven by nucleotide occupancy. The relative orientation of N and C lobes indicates a “closed” rather than “open” conformation. Par3CR3 contacts extend from a pocket beneath the ribose-binding pocket of PKCι (site 1), across the G helix (site 2) through to the activation loop, αB and αC helices of the PKCιKD-2P N lobe (site 3) (Figures 2A and 2B). A total surface area of more than 1,305 Å2 is buried within the complex, consistent with a high-affinity inhibitory interaction. The nucleotide cleft is occupied by an Mg-AMPPNP nucleotide (Figure 2C). The conserved nucleotide-coordinating lysine (K283PKCι) forms a salt bridge with the conserved αC-helix glutamate (E302PKCι) side chain found in many active kinase conformers (Kornev et al., 2008). The terminal γ-phosphate of AMPPNP is not observed in the structure, consistent with AMPPNP being rapidly hydrolyzed under the crystallization conditions (see Experimental Procedures). A magnesium ion, equivalent to Mg2 of PKA, is present, bridging both the α and β phosphates of AMPPNP (Adams and Taylor, 1993, Zheng et al., 1993). The Mg1 ion is not present, as frequently found in ADP-complexed AGC kinase structures.

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