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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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Par3/Baz CR3-Mediated Inhibition of aPKC In Vitro(A) Domain structure of Par3 and PKCι and location of key phosphorylation sites in each. For more detail on aPKC and Par3/Baz subcellular localization and how aPKC phosphorylation of Par3/Baz switches Par3/Baz localization from the apical membrane to AJs, see Figure S1.(B) Sequence alignment of human Par3 CR3 region with Par1 highlighting known phosphorylation sites (red).(C) Par3CR3 inhibits PKCιKD-2P catalytic activity in an in vitro kinase assay, whereas a Par1-derived peptide is a substrate.(D) The IC50 curves for Par3CR3.(E) Affinity of fluorescein-labeled Par3CR3 for PKCιKD-2P measured by fluorescence anisotropy.RFU, relative fluorescence units; WT, wild-type. Data are plotted as mean ± SEM. See also Figure S2 for purification and further characterization of PKCιKD-2P.
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fig1: Par3/Baz CR3-Mediated Inhibition of aPKC In Vitro(A) Domain structure of Par3 and PKCι and location of key phosphorylation sites in each. For more detail on aPKC and Par3/Baz subcellular localization and how aPKC phosphorylation of Par3/Baz switches Par3/Baz localization from the apical membrane to AJs, see Figure S1.(B) Sequence alignment of human Par3 CR3 region with Par1 highlighting known phosphorylation sites (red).(C) Par3CR3 inhibits PKCιKD-2P catalytic activity in an in vitro kinase assay, whereas a Par1-derived peptide is a substrate.(D) The IC50 curves for Par3CR3.(E) Affinity of fluorescein-labeled Par3CR3 for PKCιKD-2P measured by fluorescence anisotropy.RFU, relative fluorescence units; WT, wild-type. Data are plotted as mean ± SEM. See also Figure S2 for purification and further characterization of PKCιKD-2P.

Mentions: The human Par3 conserved region 3 (CR3, covering residues 816–834, defined hereafter as Par3CR3) is able to bind to PKCι (Nagai-Tamai et al., 2002) and contains a phospho-acceptor site (P site) at residue serine 827 known to be phosphorylated by PKCι (Figures 1A and 1B). To characterize its interaction with PKCι we purified a “primed” active form of the human PKC-iota kinase domain (referred to as PKCιKD-2P) and a partially primed low-activity form (referred to as PKCιKD-1P), referring to the status of the two “priming” phosphorylation sites at pT412 and pT564 (Figures 1A and S2A–S2C). We then probed how efficiently they were able to phosphorylate Par3CR3. Surprisingly, we found that Par3CR3 strongly inhibited the catalytic activity of PKCιKD-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 μM. In contrast, peptides from other known aPKC substrates such as Par1 were efficiently phosphorylated and were unable to inhibit (Figures 1B–1D). Using a fluorescence anisotropy assay, we found that the Par3CR3 binds to PKCιKD-2P with submicromolar affinity (KD of 0.47 ± 0.09 μM), as does an S827A mutant (KD of 0.97 ± 0.07 μM) (Figure 1E). PKCιKD-2P is a good surrogate for an activated Par complex containing Par6-PKCι-Cdc42 complex that exhibits high activity in vitro and is also potently inhibited by Par3CR3 (data not shown). In contrast, PKCιKD-1P was not inhibited to the same extent and had a much lower affinity for Par3CR3 (compare Figures 1D, S2D, and S2E). We conclude that a high-affinity Par3CR3 targets PKCιKD-2P and inhibits its catalytic activity.


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Par3/Baz CR3-Mediated Inhibition of aPKC In Vitro(A) Domain structure of Par3 and PKCι and location of key phosphorylation sites in each. For more detail on aPKC and Par3/Baz subcellular localization and how aPKC phosphorylation of Par3/Baz switches Par3/Baz localization from the apical membrane to AJs, see Figure S1.(B) Sequence alignment of human Par3 CR3 region with Par1 highlighting known phosphorylation sites (red).(C) Par3CR3 inhibits PKCιKD-2P catalytic activity in an in vitro kinase assay, whereas a Par1-derived peptide is a substrate.(D) The IC50 curves for Par3CR3.(E) Affinity of fluorescein-labeled Par3CR3 for PKCιKD-2P measured by fluorescence anisotropy.RFU, relative fluorescence units; WT, wild-type. Data are plotted as mean ± SEM. See also Figure S2 for purification and further characterization of PKCιKD-2P.
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fig1: Par3/Baz CR3-Mediated Inhibition of aPKC In Vitro(A) Domain structure of Par3 and PKCι and location of key phosphorylation sites in each. For more detail on aPKC and Par3/Baz subcellular localization and how aPKC phosphorylation of Par3/Baz switches Par3/Baz localization from the apical membrane to AJs, see Figure S1.(B) Sequence alignment of human Par3 CR3 region with Par1 highlighting known phosphorylation sites (red).(C) Par3CR3 inhibits PKCιKD-2P catalytic activity in an in vitro kinase assay, whereas a Par1-derived peptide is a substrate.(D) The IC50 curves for Par3CR3.(E) Affinity of fluorescein-labeled Par3CR3 for PKCιKD-2P measured by fluorescence anisotropy.RFU, relative fluorescence units; WT, wild-type. Data are plotted as mean ± SEM. See also Figure S2 for purification and further characterization of PKCιKD-2P.
Mentions: The human Par3 conserved region 3 (CR3, covering residues 816–834, defined hereafter as Par3CR3) is able to bind to PKCι (Nagai-Tamai et al., 2002) and contains a phospho-acceptor site (P site) at residue serine 827 known to be phosphorylated by PKCι (Figures 1A and 1B). To characterize its interaction with PKCι we purified a “primed” active form of the human PKC-iota kinase domain (referred to as PKCιKD-2P) and a partially primed low-activity form (referred to as PKCιKD-1P), referring to the status of the two “priming” phosphorylation sites at pT412 and pT564 (Figures 1A and S2A–S2C). We then probed how efficiently they were able to phosphorylate Par3CR3. Surprisingly, we found that Par3CR3 strongly inhibited the catalytic activity of PKCιKD-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 μM. In contrast, peptides from other known aPKC substrates such as Par1 were efficiently phosphorylated and were unable to inhibit (Figures 1B–1D). Using a fluorescence anisotropy assay, we found that the Par3CR3 binds to PKCιKD-2P with submicromolar affinity (KD of 0.47 ± 0.09 μM), as does an S827A mutant (KD of 0.97 ± 0.07 μM) (Figure 1E). PKCιKD-2P is a good surrogate for an activated Par complex containing Par6-PKCι-Cdc42 complex that exhibits high activity in vitro and is also potently inhibited by Par3CR3 (data not shown). In contrast, PKCιKD-1P was not inhibited to the same extent and had a much lower affinity for Par3CR3 (compare Figures 1D, S2D, and S2E). We conclude that a high-affinity Par3CR3 targets PKCιKD-2P and inhibits its catalytic activity.

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