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

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.


Reducing the Par3CR3 Affinity for PKCιKD-2P Promotes Efficient CR3 Phosphorylation In Vitro(A) Summary table of kcat, KD, and KM constants between various Par3CR3 mutants and PKCιKD-2P. Data are presented as mean ± SEM. n.d., not determined.(B) Binding curves for Par3CR3 and various Par3CR3 mutants determined by fluorescence polarization (color coded as in A).(C) PKCιKD-2P catalytic activity kinetic rate constants for Par3CR3 and various Par3CR3 mutants (color coded as in A). For further details of other inhibitory peptides similar to Par3CR3, see Figure S5.(D) Co-immunoprecipitation (IP) of full-length Myc-Par3 or mutants (Par3-A-X-A and Par3-A (S827A)) and GFP-PKCι from HCT-116 cells shows that the F-X-R to A-X-A mutation dramatically reduces the interaction.(E) Co-immunoprecipitation of GFP-PKCι or GFP-PKCι-D/D with Myc-Par3 also severely impairs the interaction. GFP-PKCι-D/D is a mutant replacing residues D330/D373 that interact with the F-X-R motif by alanine.(F) Immunoblot (IB) using a phospho-S827-specific antibody indicates that Par3 and Par3 A-X-A mutant (but not Par3-A) are phosphorylated in HCT-116 cells.For details showing evidence of phosphorylation of A-X-A Baz mutant, see Figure S6.
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fig5: Reducing the Par3CR3 Affinity for PKCιKD-2P Promotes Efficient CR3 Phosphorylation In Vitro(A) Summary table of kcat, KD, and KM constants between various Par3CR3 mutants and PKCιKD-2P. Data are presented as mean ± SEM. n.d., not determined.(B) Binding curves for Par3CR3 and various Par3CR3 mutants determined by fluorescence polarization (color coded as in A).(C) PKCιKD-2P catalytic activity kinetic rate constants for Par3CR3 and various Par3CR3 mutants (color coded as in A). For further details of other inhibitory peptides similar to Par3CR3, see Figure S5.(D) Co-immunoprecipitation (IP) of full-length Myc-Par3 or mutants (Par3-A-X-A and Par3-A (S827A)) and GFP-PKCι from HCT-116 cells shows that the F-X-R to A-X-A mutation dramatically reduces the interaction.(E) Co-immunoprecipitation of GFP-PKCι or GFP-PKCι-D/D with Myc-Par3 also severely impairs the interaction. GFP-PKCι-D/D is a mutant replacing residues D330/D373 that interact with the F-X-R motif by alanine.(F) Immunoblot (IB) using a phospho-S827-specific antibody indicates that Par3 and Par3 A-X-A mutant (but not Par3-A) are phosphorylated in HCT-116 cells.For details showing evidence of phosphorylation of A-X-A Baz mutant, see Figure S6.

Mentions: Our results suggested that Par3 CR3 arms flanking the consensus PKC phosphorylation site cooperate to inhibit PKCι. To probe the individual contributions of each arm, we characterized Par3CR3 substitutions at critical contact residues in the affinity arm and the inhibitory arm for their impact on Par3CR3 affinity for PKCι and ability inhibit kinase activity. Two mutants were prepared: first, substitution of F-Q-R to A-Q-A in the site 1 affinity arm, referred to as A-X-A hereafter; and second, substitution of K-R-T to A-A-T of the site 3 inhibitory arm, referred to as A-A-T. Consistent with our crystal structure, either A-X-A or A-A-T mutation within Par3CR3 markedly reduce the CR3-binding affinity for PKCιKD-2P, but without abolishing the interaction entirely (Figures 5A and 5B). A phospho-S827Par3 peptide representing the PKCι reaction product bound poorly, with affinity two orders of magnitude lower than in Par3CR3 (Figures 5A and 5B).


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Reducing the Par3CR3 Affinity for PKCιKD-2P Promotes Efficient CR3 Phosphorylation In Vitro(A) Summary table of kcat, KD, and KM constants between various Par3CR3 mutants and PKCιKD-2P. Data are presented as mean ± SEM. n.d., not determined.(B) Binding curves for Par3CR3 and various Par3CR3 mutants determined by fluorescence polarization (color coded as in A).(C) PKCιKD-2P catalytic activity kinetic rate constants for Par3CR3 and various Par3CR3 mutants (color coded as in A). For further details of other inhibitory peptides similar to Par3CR3, see Figure S5.(D) Co-immunoprecipitation (IP) of full-length Myc-Par3 or mutants (Par3-A-X-A and Par3-A (S827A)) and GFP-PKCι from HCT-116 cells shows that the F-X-R to A-X-A mutation dramatically reduces the interaction.(E) Co-immunoprecipitation of GFP-PKCι or GFP-PKCι-D/D with Myc-Par3 also severely impairs the interaction. GFP-PKCι-D/D is a mutant replacing residues D330/D373 that interact with the F-X-R motif by alanine.(F) Immunoblot (IB) using a phospho-S827-specific antibody indicates that Par3 and Par3 A-X-A mutant (but not Par3-A) are phosphorylated in HCT-116 cells.For details showing evidence of phosphorylation of A-X-A Baz mutant, see Figure S6.
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fig5: Reducing the Par3CR3 Affinity for PKCιKD-2P Promotes Efficient CR3 Phosphorylation In Vitro(A) Summary table of kcat, KD, and KM constants between various Par3CR3 mutants and PKCιKD-2P. Data are presented as mean ± SEM. n.d., not determined.(B) Binding curves for Par3CR3 and various Par3CR3 mutants determined by fluorescence polarization (color coded as in A).(C) PKCιKD-2P catalytic activity kinetic rate constants for Par3CR3 and various Par3CR3 mutants (color coded as in A). For further details of other inhibitory peptides similar to Par3CR3, see Figure S5.(D) Co-immunoprecipitation (IP) of full-length Myc-Par3 or mutants (Par3-A-X-A and Par3-A (S827A)) and GFP-PKCι from HCT-116 cells shows that the F-X-R to A-X-A mutation dramatically reduces the interaction.(E) Co-immunoprecipitation of GFP-PKCι or GFP-PKCι-D/D with Myc-Par3 also severely impairs the interaction. GFP-PKCι-D/D is a mutant replacing residues D330/D373 that interact with the F-X-R motif by alanine.(F) Immunoblot (IB) using a phospho-S827-specific antibody indicates that Par3 and Par3 A-X-A mutant (but not Par3-A) are phosphorylated in HCT-116 cells.For details showing evidence of phosphorylation of A-X-A Baz mutant, see Figure S6.
Mentions: Our results suggested that Par3 CR3 arms flanking the consensus PKC phosphorylation site cooperate to inhibit PKCι. To probe the individual contributions of each arm, we characterized Par3CR3 substitutions at critical contact residues in the affinity arm and the inhibitory arm for their impact on Par3CR3 affinity for PKCι and ability inhibit kinase activity. Two mutants were prepared: first, substitution of F-Q-R to A-Q-A in the site 1 affinity arm, referred to as A-X-A hereafter; and second, substitution of K-R-T to A-A-T of the site 3 inhibitory arm, referred to as A-A-T. Consistent with our crystal structure, either A-X-A or A-A-T mutation within Par3CR3 markedly reduce the CR3-binding affinity for PKCιKD-2P, but without abolishing the interaction entirely (Figures 5A and 5B). A phospho-S827Par3 peptide representing the PKCι reaction product bound poorly, with affinity two orders of magnitude lower than in Par3CR3 (Figures 5A and 5B).

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.