Fig. 1
From: Nuclear fallout provides a new link between aPKC and polarized cell trafficking
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