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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.


Related in: MedlinePlus

Switching Par3/Baz from Apical to Junctional In Vivo(A) GFP-tagged Par3/Baz (green) localizes to the apical domain (marked by aPKC in red) and also to AJs.(B) Phosphomimic GFP-tagged Par3/Baz S980E (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(C) Low-affinity GFP-tagged Par3/Baz F-X-R to A-X-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(D) Low-affinity GFP-tagged Par3/Baz K-H to A-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(E) Phospho-mutant GFP-tagged Par3/Baz S980A mutant (green) co-localizes apically with aPKC (red) and also partially disrupts cell polarity, consistent with its inhibitory function. See also Figure S7 for evidence that Baz co-localizes with aPKC in the absence of kinase activity.(F) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the F-X-R to A-X-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(G) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the K-H to A-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(H) The double mutant K-H to A-A and F-X-R to A-X-A localizes primarily to AJs.(I) Apical section of GFP-BazAXA expressing follicle cell epithelium, showing junctional localization.(J–M) Apical section of GFP-BazS980A (J) expressing follicle cell epithelium, showing mislocalization to the apical surface. Apical sections of (K) GFP-BazAXA S980A-, (L) GFP-BazAA S980A-, and (M) GFP-BazAA AXA-expressing follicle cell epithelium, showing restoration of junctional localization.(N) Non-overlapping punctate localization of GFP-BazAXA AA (green) with aPKC (red).DAPI staining is shown in blue in (A)–(H) and (N). GFP-tagged Par3/Baz is shown in (A′)–(H′).
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fig6: Switching Par3/Baz from Apical to Junctional In Vivo(A) GFP-tagged Par3/Baz (green) localizes to the apical domain (marked by aPKC in red) and also to AJs.(B) Phosphomimic GFP-tagged Par3/Baz S980E (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(C) Low-affinity GFP-tagged Par3/Baz F-X-R to A-X-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(D) Low-affinity GFP-tagged Par3/Baz K-H to A-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(E) Phospho-mutant GFP-tagged Par3/Baz S980A mutant (green) co-localizes apically with aPKC (red) and also partially disrupts cell polarity, consistent with its inhibitory function. See also Figure S7 for evidence that Baz co-localizes with aPKC in the absence of kinase activity.(F) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the F-X-R to A-X-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(G) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the K-H to A-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(H) The double mutant K-H to A-A and F-X-R to A-X-A localizes primarily to AJs.(I) Apical section of GFP-BazAXA expressing follicle cell epithelium, showing junctional localization.(J–M) Apical section of GFP-BazS980A (J) expressing follicle cell epithelium, showing mislocalization to the apical surface. Apical sections of (K) GFP-BazAXA S980A-, (L) GFP-BazAA S980A-, and (M) GFP-BazAA AXA-expressing follicle cell epithelium, showing restoration of junctional localization.(N) Non-overlapping punctate localization of GFP-BazAXA AA (green) with aPKC (red).DAPI staining is shown in blue in (A)–(H) and (N). GFP-tagged Par3/Baz is shown in (A′)–(H′).

Mentions: If the affinity of the Par3/Baz-aPKC interaction essentially determines the localization of Par3/Baz in epithelial cells, then the Par3CR3 substitutions within each arm (A-X-A or A-A-T mutants) characterized in vitro should affect the localization of Par3/Baz in vivo. To test this prediction, we mutated the CR3 region of full-length GFP-tagged Drosophila Baz in the F-X-R motif to A-X-A or the K-H-T motif to A-A-T and examined their apical domain or AJ localization in vivo. In the follicular epithelium, GFP-tagged wild-type Baz (GFP-Baz) co-localizes with aPKC at the apical membrane and also localizes to AJs (Figure 6A). Phospho-Baz is known to localize to AJs (Morais-de-Sa et al., 2010), and a GFP-tagged phosphomimic version of Baz (GFP-Baz S980E) expectedly fails to co-localize with aPKC at the apical membrane but instead localizes to AJs (Figure 6B) (Morais-de-Sa et al., 2010, Walther and Pichaud, 2010). Both the GFP-Baz A-X-A and A-A-T mutant localize similarly to the phospho-mimetic (Figures 6C, 6D, and 6I), consistent with the view that lowering affinity (as observed in cells; Figure 5B) and/or removing inhibitory elements from CR3 induces phosphorylation of Baz (as observed in vitro; Figure 5) and therefore results in its localization to AJs rather than stable Par complex formation at the apical membrane.


aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization
Switching Par3/Baz from Apical to Junctional In Vivo(A) GFP-tagged Par3/Baz (green) localizes to the apical domain (marked by aPKC in red) and also to AJs.(B) Phosphomimic GFP-tagged Par3/Baz S980E (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(C) Low-affinity GFP-tagged Par3/Baz F-X-R to A-X-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(D) Low-affinity GFP-tagged Par3/Baz K-H to A-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(E) Phospho-mutant GFP-tagged Par3/Baz S980A mutant (green) co-localizes apically with aPKC (red) and also partially disrupts cell polarity, consistent with its inhibitory function. See also Figure S7 for evidence that Baz co-localizes with aPKC in the absence of kinase activity.(F) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the F-X-R to A-X-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(G) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the K-H to A-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(H) The double mutant K-H to A-A and F-X-R to A-X-A localizes primarily to AJs.(I) Apical section of GFP-BazAXA expressing follicle cell epithelium, showing junctional localization.(J–M) Apical section of GFP-BazS980A (J) expressing follicle cell epithelium, showing mislocalization to the apical surface. Apical sections of (K) GFP-BazAXA S980A-, (L) GFP-BazAA S980A-, and (M) GFP-BazAA AXA-expressing follicle cell epithelium, showing restoration of junctional localization.(N) Non-overlapping punctate localization of GFP-BazAXA AA (green) with aPKC (red).DAPI staining is shown in blue in (A)–(H) and (N). GFP-tagged Par3/Baz is shown in (A′)–(H′).
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Related In: Results  -  Collection

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fig6: Switching Par3/Baz from Apical to Junctional In Vivo(A) GFP-tagged Par3/Baz (green) localizes to the apical domain (marked by aPKC in red) and also to AJs.(B) Phosphomimic GFP-tagged Par3/Baz S980E (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(C) Low-affinity GFP-tagged Par3/Baz F-X-R to A-X-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(D) Low-affinity GFP-tagged Par3/Baz K-H to A-A mutant (green) is largely excluded from the apical domain (marked by aPKC in red) and localizes to AJs.(E) Phospho-mutant GFP-tagged Par3/Baz S980A mutant (green) co-localizes apically with aPKC (red) and also partially disrupts cell polarity, consistent with its inhibitory function. See also Figure S7 for evidence that Baz co-localizes with aPKC in the absence of kinase activity.(F) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the F-X-R to A-X-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(G) Phospho-mutant GFP-tagged Par3/Baz S980A that also carries the K-H to A-A mutation (green) fails to co-localize with aPKC (red) and instead localizes to AJs.(H) The double mutant K-H to A-A and F-X-R to A-X-A localizes primarily to AJs.(I) Apical section of GFP-BazAXA expressing follicle cell epithelium, showing junctional localization.(J–M) Apical section of GFP-BazS980A (J) expressing follicle cell epithelium, showing mislocalization to the apical surface. Apical sections of (K) GFP-BazAXA S980A-, (L) GFP-BazAA S980A-, and (M) GFP-BazAA AXA-expressing follicle cell epithelium, showing restoration of junctional localization.(N) Non-overlapping punctate localization of GFP-BazAXA AA (green) with aPKC (red).DAPI staining is shown in blue in (A)–(H) and (N). GFP-tagged Par3/Baz is shown in (A′)–(H′).
Mentions: If the affinity of the Par3/Baz-aPKC interaction essentially determines the localization of Par3/Baz in epithelial cells, then the Par3CR3 substitutions within each arm (A-X-A or A-A-T mutants) characterized in vitro should affect the localization of Par3/Baz in vivo. To test this prediction, we mutated the CR3 region of full-length GFP-tagged Drosophila Baz in the F-X-R motif to A-X-A or the K-H-T motif to A-A-T and examined their apical domain or AJ localization in vivo. In the follicular epithelium, GFP-tagged wild-type Baz (GFP-Baz) co-localizes with aPKC at the apical membrane and also localizes to AJs (Figure 6A). Phospho-Baz is known to localize to AJs (Morais-de-Sa et al., 2010), and a GFP-tagged phosphomimic version of Baz (GFP-Baz S980E) expectedly fails to co-localize with aPKC at the apical membrane but instead localizes to AJs (Figure 6B) (Morais-de-Sa et al., 2010, Walther and Pichaud, 2010). Both the GFP-Baz A-X-A and A-A-T mutant localize similarly to the phospho-mimetic (Figures 6C, 6D, and 6I), consistent with the view that lowering affinity (as observed in cells; Figure 5B) and/or removing inhibitory elements from CR3 induces phosphorylation of Baz (as observed in vitro; Figure 5) and therefore results in its localization to AJs rather than stable Par complex formation at the apical membrane.

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