Phosphorylation‐dependent Akt–Inversin interaction at the basal body of primary cilia

• DOI: 10.15252/embj.201593003
• Publication Date: 2016-05-25T00:40:03Z
• Authors: Futoshi Suizu; Noriyuki Hirata; Kohki Kimura; Tatsuma Edamura; Tsutomu Tanaka; Satoko Ishigaki; Thoria Donia; Hiroko Noguchi; Toshihiko Iwanaga; Masayuki Noguchi
• Keywords: Basal (medicine); Basal body; Primary (astronomy)

Article24 May 2016Open Access Transparent process Phosphorylation-dependent Akt–Inversin interaction at the basal body of primary cilia Futoshi Suizu Division Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search more papers by this author Noriyuki Hirata Kohki Kimura Tatsuma Edamura Tsutomu Tanaka Satoko Ishigaki Thoria Donia Chemistry Department, Faculty Science, Tanta Tanta, Egypt Hiroko Noguchi Department Pathology, Teine Keijinkai Hospital, Toshihiko Iwanaga Laboratory Histology and Cytology, University Graduate School Masayuki Corresponding Author orcid.org/0000-0002-1787-2724 Information Suizu1, Hirata1, Kimura1, Edamura1, Tanaka1, Ishigaki1, Donia2, Noguchi3, Iwanaga4 1 1Division 2Chemistry 3Department 4Laboratory *Corresponding author. Tel: [phone]; Fax: 7826; E-mail: [email protected] The EMBO Journal (2016)35:1346-1363https://doi.org/10.15252/embj.201593003 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A cilium is microtubule-based sensory organelle that plays an important role in human development disease. However, regulation Akt its ciliary has not been demonstrated. Using yeast two-hybrid screening, we demonstrate Inversin (INVS) interacts with Akt. Mutation INVS gene causes nephronophthisis type II (NPHP2), autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show via C-terminus. In vitro kinase phosphorylates amino acids 864–866 are required only interaction, but also dimerization. Co-localization phosphorylated form augmented PDGF-AA. Akt-null MEF cells as well siRNA-mediated inhibition attenuated growth, which was reversed reintroduction. Mutant phosphodead- or NPHP2-related truncated INVS, lack phosphorylation sites, suppress cell growth exhibit distorted lumen formation misalignment spindle axis during division. Further studies will be elucidating functional interactions Akt–INVS identifying molecular mechanisms underlying NPHP2. Synopsis Cilia organelles play roles kidney cilial protein nephropathy (nephronophthisis II; NPHP2), accordingly, found associate nephrocystin 3 (NPHP3) microtubule cytoskeleton. Here, shown regulate physiology renal integrity body. directly INVS. conserved Akt, homodimerization. p-Akt co-localize base centrosome, recruitment stimulated PDGFRα/Akt signaling. MDCK expressing mutant lacks site impaired ciliogenesis division, leading suppressed tubular lumina. Introduction Primary (MT)-based arise from surrounded pericentriolar material extend surface (where mother centriole located) arrest most vertebrate (Nigg Raff, 2009; Hildebrandt et al, 2011) (Suizu 2016). Based on their MT components, classified motile (9 + 2) 0) (Marshall, 2008; Gerdes 2009). physiological importance established given disruption malfunction results variety diseases called "ciliopathy", includes cystic diseases, hydrocephalus, blindness, obesity, polydactyly, diabetes, cognitive impairment, developmental disorders (Otto 2003; Zariwala 2007; Adams Marshall, Nigg Tory 2011). serine–threonine major downstream target PI3K, cancer pathogenesis inhibiting apoptosis maintaining metabolism autophagy. Three isoforms exist mammalian (Brazil 2004; Manning Cantley, 2014). Through biological targets, regulates wide range cellular processes, survival, cycle, proliferation, cytoskeletal organization, vesicle trafficking, glucose transport, platelet function (Noguchi 2007). Therefore, contributes cancer, intolerance, schizophrenia, viral infections, autoimmune involvement signal transduction poorly characterized. study, (INVS), highly ankyrin repeats IQ domains, specifically bind both Akt2 Akt3 screening (Mochizuki 1998; Morgan 2002b; Eley 2004). Mutations result disease progresses end-stage failure early infancy occasionally associated situs inversus Saunier 2005; Badano 2006; Cystic caused amazingly broad array genetic mutations manipulations dysfunction suggested (Oh Katsanis, 2012). Altered signaling disoriented division tubules, resulting cyst (Happe localizes epithelial nodal monocilia mouse embryos within so-called compartment located proximal part (Morgan Watanabe Shiba During anaphase, relocalizes poles, coinciding translocation HEF1 (human enhancer filamentation) centrosome (Eley Despite identification increasing number interacting proteins, fully clarified. Dishevelled (Dvl), activates PCP (planar polarity) signaling, simultaneously downregulates canonical Wnt targeting Dvl proteasome-mediated degradation (Simons 2005). anaphase-promoting complex 2 (APC2) through D-box motif, suggesting it involved cycle 2002b). co-precipitates N-cadherin catenins, β-catenin, calmodulin polarized (Yasuhiko 2001; Nurnberger 2002; coordinates ANKS6 NPHP2 (Hoff 2013). Moreover, Aurora A, thereby activation, consequently interferes HDAC6-mediated disassembly (Mergen implicated various control events, sonic hedgehog (Shh) (Corbit 2005), (Benzing Lancaster 2011), TGF-β (Clement 2013a), platelet-derived factor (PDGF)-mediated (Schneider PDGF receptor alpha (PDGFRα) fibroblast migration pathway 2013b). Consistent observation, factors, PDGF, mediating signals 2007, These findings support possible cilia. severe cysts understood current portion cilia, PDGF-AA-dependent manner. acid residues 864–866. either phosphorylation-defective mutants sites into (Madin–Darby canine kidney) inhibits proliferation aberrant orientation exhibiting formation. observations indicate between significance dysregulation may abnormal Results associates We conducted screens using baits. One molecule bound partial coding sequence (860–1,007) Interestingly, partially overlapping (755–955) interact Akt3. To confirm cells, performed co-immunoprecipitation assays. Flag-INVS interacted HA-Akt1, HA-PDK1 HA-PrKA, supporting specificity observed (Fig 1A). Since next examined each three isoforms. HA-Akt2, HA-Akt3 1B). endogenous determined HEK293 expressed levels 1C). Figure 1. HA-Akt1 (lanes 4–6), 7–9) HA-PrKA 10–12) HEK293T cells. Expression PDK1, PrKA, were similar, immunoblotting (HA: anti-HA antibody; F: anti-Flag C: antibody). Flag-tagged HA-tagged Akt1 7–9), Expressions isoform similar. co-immunoprecipitated (lane 2), endogenously expressed. determine domain responsible subfragments generated. C-terminus 10–12). Similar fragment immunoprecipitated antibody. Schematic showing structure domains full-length fragmented expression vectors (N-terminal: 1–421, intermediate domain: 422–675, C-terminal: 676–1065) used study. Intermediate 11–13) C-terminal 14–16) fragments Data information: presented (A–E) representative least two independent experiments. Download figure PowerPoint identify generated HA-Akt additional mediated middle-to-C-terminal (Int INVS: 421–675, 676–1,065) 1D E). T864, S865, T866 PDGFRα mitosis present (Wakefield Zhu whether might direct substrate (IVK) employed multiple fragments, able phosphorylate WT, 1–970, 1–898 failed 1–670 2A Appendix Fig S1A). 2. novel indicated recombinant proteins 1–4) IVK. WT 5), 1–970 6), 7) 8) arrows (right side) position 1-670 4), 8). 1–10) 6) other (1–822, 1–746, 1–670, lanes 8, 9, 10, respectively), detected phospho-Akt IVK, confirmed 675–1065 675–822 675–746 Amino alignment 824–898. Motif scan analysis Scansite3 database identified acids, T866, putative targets. Purified 3A (top panel, 1–4). (824–898, middle lane 3), (middle 9), 12) 15) 3A, ∆727–896, 1–857, 1–822 16, 11, 13, 14, all lacked T864/S865/T866 acid. Next, utilized deletion further dissecting IVK 1–822, 2B). site(s) contrast did these results, 675–1,065 fragment, 2C). Together, region 823 898. program Scansite 2009), consecutive (T864, T866) targets 2D). serine threonine species Pan troglodytes, Canis lupus familiaris, Bos taurus, Oryctolagus cuniculus, vivo (Appendix S1B). Recombinant wild-type (WT) triple-alanine-substitution 864, 865, 866 (hereafter designated "3A") 824–898 subfragment (824–898), 2E). Importantly, 1–898, subfragment, 727–896, subfragments, 2F). one several serine/threonine T864/S865/T866. controls migration, differentiation, activity specialized mesenchymal migratory types, adult animal (Hoch Soriano, Schneider 2005, 2010; Christensen Clement organize (from centriole) where activated constitutively (Zhu development. confocal microscopy, active (phospho-S473 Akt) localized centrosome-like dividing 3A). previous reports, (Shiba 2009) 3B). co-localized γ-tubulin, marker, confluent 3C). Phosphorylated anti-phospho-Akt antibody immunoblots (see 3D). Upon PDGF-AA stimulation, appeared same pocket. PI3K inhibitor (LY294002) inhibited phospho-S473 localization pocket S2B-E). 3. Both Confocal microscopy shows (arrows) (right-side panels higher magnification). (Inversin compartment, right-side co-localizes marker (arrows). Anti-phospho-Akt substrates stain positively (arrows), anti-acetylated tubulin staining Silver-intensified immunogold electron (left-side panels) presence (white arrows). representation view microscope. dissect precise base, silver-intensified method microscopy. Our showed body, close pocket, after treatment 3E F). accumulated prior S2A). stimulates co-localization known mediate intracellular stimulation affected resulted time-dependent increase 4A B). 4. increases enhances Quantification (green) (red) time points revealed increase. measured counting yellow pixels (co-localized area) Imaris software (Bitplane AG). means ± SE (n = 31 0, n 35 min, 33 min). experiments analyzed similar results. Statistical Student's t-test. preferentially binds (second 1) phospho-mimetic (T308D/S473D, fails forms (T308A/S473A, 3). comparison 2, untreated PDGF-AA-stimulated conditions, respectively). 3A-INVS, under serum-starved condition panels, lower magnification), translocate demonstrating EGFP-INVS γ-tubulin hTERT-RPE1 28). Fluorescence intensities (green), acetylated α-tubulin (blue), along line (a-b) plotted underneath. affects assays, (T308D/S473D), phosphorylation-deficient (T308A/S473A) 4C). examine activation PDGF-AA, mediates As expected, clearly administration 4D). how (WT vs. report conditions 4E, left-side panels). After however, relocalized around translocated stimulation. thus far supported hypothesis played growth. context. treatment, induced 5A, top panel). could LY294002, MK2206, GSK690693 inhibitors indicating indeed An S473 T308 due feedback mechanism (Okuzumi obtained serum S3A) COS-7 S3B). 5. Decrease GSK690693, 3, 4, 5, Myr-Akt, together quantified. can Myr-Akt. exhibited weaker compared compare phospho-defective mutant, dimerization below each. Length knockdown Akt1/2. Ciliary length decreased siRNA. longitudinal (red), siRNA-transfected (green: EGFP positive) 175 siRNA siRNA, 195 plus siRNA-resistant Akt). Note reintroduction (Matsuda-Lennikov 2014) rescued effect length. (bottom mean transfected 58 vector 52 mutant). co-transfected constitutive induce de