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Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

Calero-Cuenca FJ, Espinosa-Vázquez JM, Reina-Campos M, Díaz-Meco MT, Moscat J, Sotillos S - BMC Biol. (2016)

Bottom Line: Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution.We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached.

View Article: PubMed Central - PubMed

Affiliation: CABD, CSIC/JA/UPO, Campus Universidad Pablo de Olavide, Ctra. De Utrera Km. 1, Seville, 41013, Spain.

ABSTRACT

Background: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.

Results: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.

Conclusions: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

No MeSH data available.


Related in: MedlinePlus

Nuf directly interacts with and is phosphorylated by aPKC. a aPKC full-length, regulatory and kinase domain Tetra-Tag (TT) constructs used in pull-down assays. b Pull-down (PD) assay of wild-type embryonic extracts with GST or GST-Nuf. Immunoblotting of bound proteins with anti-aPKC show aPKC presence only in the Nuf-containing sample. c In vitro assay of aPKC binding to Nuf. Recombinant aPKC was incubated in vitro with recombinant purified GST or GST-Nuf. Immunoblotting with anti-aPKC shows direct binding of aPKC to Nuf. d The kinase domain of aPKC interacts with Nuf. Embryonic extracts were incubated with aPKC kinase (Flag-aPKCKD) or regulatory domain (Flag-aPKCRD) bound to magnetic beads. Immunoblotting with anti-Nuf shows Nuf only binds to the kinase domain. The observed higher molecular weight in Nuf bound to the kinase domain of aPKC is consistent with Nuf being phosphorylated by aPKC. Asterisks point to aPKC versions used in the pull-downs cross-reacting with anti-Nuf. e Nuf only binds active aPKC. Wild type kinase domain (Flag-aPKCKD) and inactive kinase version (Flag-aPKCKD-mut) were purified with magnetic beads from embryonic extracts before incubation with purified recombinant Nuf-GST. Immunoblotting of bound proteins with anti-Nuf shows interaction with the wild type kinase domain. f Amino acid sequence and schematic representation of Nuf full-length and variants fused to GST. Asterisk marks Nuf N terminal fragment end. Orange: coiled-coil domain (CBD). Pink: Rab11 binding domain (RBD). Ser155 is highlighted in red. g Autoradiography of Nuf-GST variants subjected to aPKCz phosphorylation in vitro. Only full-length and N-terminal Nuf were phosphorylated (red asterisks). Black asterisk marks auto-phosphorylated aPKC. Preference towards substrate phosphorylation caused a reduction in autophosphorylation activity of aPKC. h Nuf phosphorylation sites. In vitro aPKC kinase was unable to phosphorylate S155A. i Recombinant GST-wild-type (WT), non-phosphorylatable (S155A) or phosphomimetic (S155D) Nuf pull-down assays from embryonic extracts. Immunoblotting probed with anti-aPKC shows Nuf S155D cannot interact with aPKC. “Input” contains 10 % (b, d, i) or 5 % (c) of the extracts. b-e, g-i. Lower panels are loading controls. Blots were probed with the indicated antibodies. aPKC atypical PKC, Nuf nuclear fallout, GST glutathione S-transferase
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Fig1: Nuf directly interacts with and is phosphorylated by aPKC. a aPKC full-length, regulatory and kinase domain Tetra-Tag (TT) constructs used in pull-down assays. b Pull-down (PD) assay of wild-type embryonic extracts with GST or GST-Nuf. Immunoblotting of bound proteins with anti-aPKC show aPKC presence only in the Nuf-containing sample. c In vitro assay of aPKC binding to Nuf. Recombinant aPKC was incubated in vitro with recombinant purified GST or GST-Nuf. Immunoblotting with anti-aPKC shows direct binding of aPKC to Nuf. d The kinase domain of aPKC interacts with Nuf. Embryonic extracts were incubated with aPKC kinase (Flag-aPKCKD) or regulatory domain (Flag-aPKCRD) bound to magnetic beads. Immunoblotting with anti-Nuf shows Nuf only binds to the kinase domain. The observed higher molecular weight in Nuf bound to the kinase domain of aPKC is consistent with Nuf being phosphorylated by aPKC. Asterisks point to aPKC versions used in the pull-downs cross-reacting with anti-Nuf. e Nuf only binds active aPKC. Wild type kinase domain (Flag-aPKCKD) and inactive kinase version (Flag-aPKCKD-mut) were purified with magnetic beads from embryonic extracts before incubation with purified recombinant Nuf-GST. Immunoblotting of bound proteins with anti-Nuf shows interaction with the wild type kinase domain. f Amino acid sequence and schematic representation of Nuf full-length and variants fused to GST. Asterisk marks Nuf N terminal fragment end. Orange: coiled-coil domain (CBD). Pink: Rab11 binding domain (RBD). Ser155 is highlighted in red. g Autoradiography of Nuf-GST variants subjected to aPKCz phosphorylation in vitro. Only full-length and N-terminal Nuf were phosphorylated (red asterisks). Black asterisk marks auto-phosphorylated aPKC. Preference towards substrate phosphorylation caused a reduction in autophosphorylation activity of aPKC. h Nuf phosphorylation sites. In vitro aPKC kinase was unable to phosphorylate S155A. i Recombinant GST-wild-type (WT), non-phosphorylatable (S155A) or phosphomimetic (S155D) Nuf pull-down assays from embryonic extracts. Immunoblotting probed with anti-aPKC shows Nuf S155D cannot interact with aPKC. “Input” contains 10 % (b, d, i) or 5 % (c) of the extracts. b-e, g-i. Lower panels are loading controls. Blots were probed with the indicated antibodies. aPKC atypical PKC, Nuf nuclear fallout, GST glutathione S-transferase

Mentions: aPKC is a Ser/Thr kinase regulated by its interaction with other proteins [9]. In a Tandem Affinity Purification biochemical screen [8] to identify new aPKC partners/substrates we isolated Nuf (Fig. 1a and Additional file 1: Figure S1). GST pull-down assays using embryonic extracts and GST-Nuf recombinant protein confirm a strong interaction with endogenous aPKC (Fig. 1b). Pull-down assays of GST-Nuf and aPKC recombinant proteins indicate a direct interaction (Fig. 1c).Fig. 1


Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

Calero-Cuenca FJ, Espinosa-Vázquez JM, Reina-Campos M, Díaz-Meco MT, Moscat J, Sotillos S - BMC Biol. (2016)

Nuf directly interacts with and is phosphorylated by aPKC. a aPKC full-length, regulatory and kinase domain Tetra-Tag (TT) constructs used in pull-down assays. b Pull-down (PD) assay of wild-type embryonic extracts with GST or GST-Nuf. Immunoblotting of bound proteins with anti-aPKC show aPKC presence only in the Nuf-containing sample. c In vitro assay of aPKC binding to Nuf. Recombinant aPKC was incubated in vitro with recombinant purified GST or GST-Nuf. Immunoblotting with anti-aPKC shows direct binding of aPKC to Nuf. d The kinase domain of aPKC interacts with Nuf. Embryonic extracts were incubated with aPKC kinase (Flag-aPKCKD) or regulatory domain (Flag-aPKCRD) bound to magnetic beads. Immunoblotting with anti-Nuf shows Nuf only binds to the kinase domain. The observed higher molecular weight in Nuf bound to the kinase domain of aPKC is consistent with Nuf being phosphorylated by aPKC. Asterisks point to aPKC versions used in the pull-downs cross-reacting with anti-Nuf. e Nuf only binds active aPKC. Wild type kinase domain (Flag-aPKCKD) and inactive kinase version (Flag-aPKCKD-mut) were purified with magnetic beads from embryonic extracts before incubation with purified recombinant Nuf-GST. Immunoblotting of bound proteins with anti-Nuf shows interaction with the wild type kinase domain. f Amino acid sequence and schematic representation of Nuf full-length and variants fused to GST. Asterisk marks Nuf N terminal fragment end. Orange: coiled-coil domain (CBD). Pink: Rab11 binding domain (RBD). Ser155 is highlighted in red. g Autoradiography of Nuf-GST variants subjected to aPKCz phosphorylation in vitro. Only full-length and N-terminal Nuf were phosphorylated (red asterisks). Black asterisk marks auto-phosphorylated aPKC. Preference towards substrate phosphorylation caused a reduction in autophosphorylation activity of aPKC. h Nuf phosphorylation sites. In vitro aPKC kinase was unable to phosphorylate S155A. i Recombinant GST-wild-type (WT), non-phosphorylatable (S155A) or phosphomimetic (S155D) Nuf pull-down assays from embryonic extracts. Immunoblotting probed with anti-aPKC shows Nuf S155D cannot interact with aPKC. “Input” contains 10 % (b, d, i) or 5 % (c) of the extracts. b-e, g-i. Lower panels are loading controls. Blots were probed with the indicated antibodies. aPKC atypical PKC, Nuf nuclear fallout, GST glutathione S-transferase
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Related In: Results  -  Collection

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Fig1: Nuf directly interacts with and is phosphorylated by aPKC. a aPKC full-length, regulatory and kinase domain Tetra-Tag (TT) constructs used in pull-down assays. b Pull-down (PD) assay of wild-type embryonic extracts with GST or GST-Nuf. Immunoblotting of bound proteins with anti-aPKC show aPKC presence only in the Nuf-containing sample. c In vitro assay of aPKC binding to Nuf. Recombinant aPKC was incubated in vitro with recombinant purified GST or GST-Nuf. Immunoblotting with anti-aPKC shows direct binding of aPKC to Nuf. d The kinase domain of aPKC interacts with Nuf. Embryonic extracts were incubated with aPKC kinase (Flag-aPKCKD) or regulatory domain (Flag-aPKCRD) bound to magnetic beads. Immunoblotting with anti-Nuf shows Nuf only binds to the kinase domain. The observed higher molecular weight in Nuf bound to the kinase domain of aPKC is consistent with Nuf being phosphorylated by aPKC. Asterisks point to aPKC versions used in the pull-downs cross-reacting with anti-Nuf. e Nuf only binds active aPKC. Wild type kinase domain (Flag-aPKCKD) and inactive kinase version (Flag-aPKCKD-mut) were purified with magnetic beads from embryonic extracts before incubation with purified recombinant Nuf-GST. Immunoblotting of bound proteins with anti-Nuf shows interaction with the wild type kinase domain. f Amino acid sequence and schematic representation of Nuf full-length and variants fused to GST. Asterisk marks Nuf N terminal fragment end. Orange: coiled-coil domain (CBD). Pink: Rab11 binding domain (RBD). Ser155 is highlighted in red. g Autoradiography of Nuf-GST variants subjected to aPKCz phosphorylation in vitro. Only full-length and N-terminal Nuf were phosphorylated (red asterisks). Black asterisk marks auto-phosphorylated aPKC. Preference towards substrate phosphorylation caused a reduction in autophosphorylation activity of aPKC. h Nuf phosphorylation sites. In vitro aPKC kinase was unable to phosphorylate S155A. i Recombinant GST-wild-type (WT), non-phosphorylatable (S155A) or phosphomimetic (S155D) Nuf pull-down assays from embryonic extracts. Immunoblotting probed with anti-aPKC shows Nuf S155D cannot interact with aPKC. “Input” contains 10 % (b, d, i) or 5 % (c) of the extracts. b-e, g-i. Lower panels are loading controls. Blots were probed with the indicated antibodies. aPKC atypical PKC, Nuf nuclear fallout, GST glutathione S-transferase
Mentions: aPKC is a Ser/Thr kinase regulated by its interaction with other proteins [9]. In a Tandem Affinity Purification biochemical screen [8] to identify new aPKC partners/substrates we isolated Nuf (Fig. 1a and Additional file 1: Figure S1). GST pull-down assays using embryonic extracts and GST-Nuf recombinant protein confirm a strong interaction with endogenous aPKC (Fig. 1b). Pull-down assays of GST-Nuf and aPKC recombinant proteins indicate a direct interaction (Fig. 1c).Fig. 1

Bottom Line: Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution.We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached.

View Article: PubMed Central - PubMed

Affiliation: CABD, CSIC/JA/UPO, Campus Universidad Pablo de Olavide, Ctra. De Utrera Km. 1, Seville, 41013, Spain.

ABSTRACT

Background: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.

Results: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.

Conclusions: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

No MeSH data available.


Related in: MedlinePlus