Phosphorylation‐dependent Akt–Inversin interaction at the basal body of primary cilia
2016; Springer Nature; Volume: 35; Issue: 12 Linguagem: Inglês
10.15252/embj.201593003
ISSN1460-2075
AutoresFutoshi Suizu, Noriyuki Hirata, Kohki Kimura, Tatsuma Edamura, Tsutomu Tanaka, Satoko Ishigaki, Thoria Donia, Hiroko Noguchi, Toshihiko Iwanaga, Masayuki Noguchi,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoArticle24 May 2016Open Access Transparent process Phosphorylation-dependent Akt–Inversin interaction at the basal body of primary cilia Futoshi Suizu Futoshi Suizu Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Noriyuki Hirata Noriyuki Hirata Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Kohki Kimura Kohki Kimura Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Tatsuma Edamura Tatsuma Edamura Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Tsutomu Tanaka Tsutomu Tanaka Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Satoko Ishigaki Satoko Ishigaki Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Thoria Donia Thoria Donia Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt Search for more papers by this author Hiroko Noguchi Hiroko Noguchi Department of Pathology, Teine Keijinkai Hospital, Sapporo, Japan Search for more papers by this author Toshihiko Iwanaga Toshihiko Iwanaga Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Search for more papers by this author Masayuki Noguchi Corresponding Author Masayuki Noguchi orcid.org/0000-0002-1787-2724 Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Futoshi Suizu Futoshi Suizu Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Noriyuki Hirata Noriyuki Hirata Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Kohki Kimura Kohki Kimura Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Tatsuma Edamura Tatsuma Edamura Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Tsutomu Tanaka Tsutomu Tanaka Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Satoko Ishigaki Satoko Ishigaki Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Thoria Donia Thoria Donia Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt Search for more papers by this author Hiroko Noguchi Hiroko Noguchi Department of Pathology, Teine Keijinkai Hospital, Sapporo, Japan Search for more papers by this author Toshihiko Iwanaga Toshihiko Iwanaga Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Search for more papers by this author Masayuki Noguchi Corresponding Author Masayuki Noguchi orcid.org/0000-0002-1787-2724 Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Author Information Futoshi Suizu1, Noriyuki Hirata1, Kohki Kimura1, Tatsuma Edamura1, Tsutomu Tanaka1, Satoko Ishigaki1, Thoria Donia2, Hiroko Noguchi3, Toshihiko Iwanaga4 and Masayuki Noguchi 1 1Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan 2Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt 3Department of Pathology, Teine Keijinkai Hospital, Sapporo, Japan 4Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo, Japan *Corresponding author. Tel: +81 11 706 5069; Fax: +81 11 706 7826; E-mail: [email protected] The EMBO Journal (2016)35:1346-1363https://doi.org/10.15252/embj.201593003 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A primary cilium is a microtubule-based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two-hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show that Akt interacts with INVS via the C-terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864–866 that are required not only for Akt interaction, but also for INVS dimerization. Co-localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF-AA. Akt-null MEF cells as well as siRNA-mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodead- or NPHP2-related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt–INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2. Synopsis Cilia are sensory organelles that play important roles in human kidney development and disease. Mutation of the cilial protein Inversin (INVS) causes autosomal recessive chronic nephropathy (nephronophthisis type II; NPHP2), and accordingly, INVS has been found to associate with nephrocystin 3 (NPHP3) and microtubule cytoskeleton. Here, the kinase Akt is shown to regulate cilia physiology and renal integrity by interaction with the INVS at the basal body. Akt directly interacts with and phosphorylates INVS. INVS conserved amino acids 864–866 are required for phosphorylation by Akt, as well as INVS homodimerization. INVS and p-Akt co-localize at the ciliary base centrosome, and recruitment of INVS is stimulated by PDGFRα/Akt signaling. MDCK cells expressing a mutant INVS that lacks the phosphorylation site 864–866 exhibit impaired ciliogenesis and misalignment of spindle axis during cell division, leading to suppressed cell growth and distorted formation of tubular lumina. Introduction Primary cilia are microtubule (MT)-based sensory organelles that arise from a basal body surrounded by pericentriolar material and extend from the surface (where the mother centriole is located) during growth arrest in most vertebrate cells (Nigg & Raff, 2009; Hildebrandt et al, 2011) (Suizu et al, 2016). Based on their MT components, cilia are classified as motile (9 + 2) or primary (9 + 0) (Marshall, 2008; Gerdes et al, 2009). The physiological importance of primary cilia is well established given that disruption or malfunction of cilia results in a variety of human diseases called "ciliopathy", which includes cystic kidney diseases, hydrocephalus, blindness, obesity, polydactyly, diabetes, cognitive impairment, and developmental disorders (Otto et al, 2003; Zariwala et al, 2007; Adams et al, 2008; Marshall, 2008; Gerdes et al, 2009; Nigg & Raff, 2009; Tory et al, 2009; Hildebrandt et al, 2011). The serine–threonine kinase Akt is a major downstream target of PI3K, which plays important roles in cancer pathogenesis by directly inhibiting apoptosis and maintaining cell metabolism by autophagy. Three isoforms of Akt exist in mammalian cells (Brazil et al, 2004; Manning & Cantley, 2007; Noguchi et al, 2014). Through its biological targets, Akt regulates a wide range of cellular processes, including survival, cell cycle, proliferation, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function (Noguchi et al, 2007). Therefore, malfunction of Akt contributes to a wide variety of human diseases including cancer, glucose intolerance, schizophrenia, viral infections, and autoimmune diseases (Brazil et al, 2004; Manning & Cantley, 2007; Noguchi et al, 2014). However, the involvement of Akt in signal transduction mechanisms in primary cilia has been poorly characterized. In this study, Inversin (INVS), a ciliary protein with highly conserved ankyrin repeats and IQ domains, was found to specifically bind both Akt2 and Akt3 by yeast two-hybrid screening (Mochizuki et al, 1998; Morgan et al, 2002b; Eley et al, 2004). Mutations in the INVS gene result in nephronophthisis type II (NPHP2), an autosomal recessive cystic kidney disease that progresses to end-stage renal failure during early infancy and is occasionally associated with situs inversus (Otto et al, 2003; Saunier et al, 2005; Badano et al, 2006; Gerdes et al, 2009; Tory et al, 2009). Cystic kidney diseases are caused by an amazingly broad array of genetic mutations and manipulations (Otto et al, 2003; Zariwala et al, 2007; Adams et al, 2008; Marshall, 2008; Gerdes et al, 2009; Nigg & Raff, 2009; Tory et al, 2009; Hildebrandt et al, 2011). However, dysfunction of cilia is suggested to play a role in cystic formation (Oh & Katsanis, 2012). Altered ciliary signaling disoriented cell division in renal tubules, resulting in renal cyst formation (Happe et al, 2011). INVS localizes to the primary cilia of renal epithelial cells and to the nodal monocilia in mouse embryos within the so-called Inversin compartment that is located in the proximal part of primary cilia (Morgan et al, 2002b; Watanabe et al, 2003; Shiba et al, 2009). During anaphase, INVS relocalizes to the spindle poles, coinciding with the translocation of HEF1 (human enhancer of filamentation) to the centrosome (Eley et al, 2004). Despite the identification of an increasing number of interacting proteins, the physiological function of INVS has not been fully clarified. INVS interacts with Dishevelled (Dvl), activates PCP (planar cell polarity) signaling, and simultaneously downregulates canonical Wnt signaling by targeting Dvl for proteasome-mediated degradation (Simons et al, 2005). INVS interacts with anaphase-promoting complex 2 (APC2) through its D-box motif, suggesting that it is involved in cell cycle regulation (Morgan et al, 2002b). INVS co-precipitates with N-cadherin and catenins, including β-catenin, as well as with calmodulin in polarized epithelial cells (Yasuhiko et al, 2001; Morgan et al, 2002b; Nurnberger et al, 2002; Saunier et al, 2005). INVS also coordinates with ANKS6 during the pathogenesis of NPHP2 (Hoff et al, 2013). Moreover, INVS interacts with Aurora A, thereby inhibiting phosphorylation and activation, which consequently interferes with HDAC6-mediated cilia disassembly (Mergen et al, 2013). Primary cilia are also implicated in the regulation of various signal transduction mechanisms that control a wide variety of cellular events, including sonic hedgehog (Shh) (Corbit et al, 2005), Wnt (Benzing et al, 2007; Lancaster et al, 2011), TGF-β (Clement et al, 2013a), and platelet-derived growth factor (PDGF)-mediated cell signaling (Schneider et al, 2005). PDGF receptor alpha (PDGFRα) signaling in the primary cilium regulates fibroblast migration via the Akt signaling pathway (Clement et al, 2013b). Consistent with this observation, Akt activates a number of growth factors, including PDGF, for mediating downstream signals (Noguchi et al, 2007, 2014). These findings support a possible functional involvement of Akt in the signal transduction of primary cilia. However, the molecular mechanisms underlying the role of INVS in the formation of severe renal cysts in NPHP2 are not fully understood (Otto et al, 2003; Tory et al, 2009). In the current study, we demonstrate that INVS, located at the basal portion of the primary cilia, interacts with Akt in a PDGF-AA-dependent manner. Akt also phosphorylates INVS at amino acid residues 864–866. Introduction of either phosphorylation-defective or NPHP2-related mutants of INVS that lack the Akt phosphorylation sites into MDCK (Madin–Darby canine kidney) cells inhibits cell proliferation with aberrant spindle axis orientation during cell division, thereby exhibiting distorted lumen formation. These observations indicate that the interaction between Akt and INVS is of biological significance for primary cilia and dysregulation of this interaction may result in abnormal cyst formation in NPHP2. Results INVS associates specifically with Akt in mammalian cells We conducted yeast two-hybrid screens using human Akt2 and Akt3 as baits. One molecule that bound specifically to Akt2 was a partial coding sequence (860–1,007) of human INVS. Interestingly, a partially overlapping coding sequence of INVS (755–955) was also found to interact with Akt3. To confirm the interaction of INVS with Akt in mammalian cells, we performed co-immunoprecipitation assays. Flag-INVS interacted with HA-Akt1, but not with HA-PDK1 or HA-PrKA, supporting the specificity of the observed interaction (Fig 1A). Since INVS was found to interact with both Akt2 and Akt3 in yeast two-hybrid screening, we next examined the specificity of the interaction for each of the three Akt isoforms. Flag-INVS interacted with HA-Akt1, HA-Akt2, and HA-Akt3 in co-immunoprecipitation assays in mammalian cells (Fig 1B). The interaction of endogenous Akt with INVS was also determined in HEK293 cells that expressed INVS at endogenous levels (Fig 1C). Figure 1. INVS specifically associates with Akt in mammalian cells Flag-INVS interacted with HA-Akt1 (lanes 4–6), but not with HA-PDK1 (lanes 7–9) or HA-PrKA (lanes 10–12) in HEK293T cells. Expression levels of HA-Akt1, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). Flag-tagged INVS interacted with HA-tagged Akt1 (lanes 4–6), Akt2 (lanes 7–9), and Akt3 (lanes 10–12) in co-immunoprecipitation assays. Expressions of each Akt isoform and INVS were similar. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells (lane 2), in which INVS is endogenously expressed. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 10–12). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-terminal: 1–421, intermediate domain: 422–675, and C-terminal: 676–1065) used in the current study. Intermediate (lanes 11–13) and C-terminal (lanes 14–16) fragments of INVS were important for interaction with Akt. Data information: The results presented (A–E) are representative of at least two independent experiments. Download figure Download PowerPoint To identify the protein domains required for the Akt–INVS interaction, we generated subfragments of HA-Akt and Flag-INVS for additional co-immunoprecipitation assays. The interaction between HA-Akt and Flag-INVS was mediated through the C-terminal kinase domain of Akt and the middle-to-C-terminal portion of INVS (Int INVS: 421–675, and C-terminal INVS: 676–1,065) (Fig 1D and E). Akt phosphorylates INVS at T864, S865, and T866 in vitro PDGFRα signaling in the primary cilium regulates fibroblast migration via Akt signal transduction mechanisms (Clement et al, 2013b). Akt is phosphorylated during mitosis and is present in the centrosome and basal body (Wakefield et al, 2003; Zhu et al, 2009). Therefore, we next examined whether INVS might be a direct substrate of Akt. Using in vitro Akt kinase assays (IVK) that employed multiple INVS fragments, Akt was able to phosphorylate WT, 1–970, and 1–898 INVS, but failed to phosphorylate the 1–670 fragment of INVS (Fig 2A and Appendix Fig S1A). Figure 2. INVS is a novel substrate of Akt The indicated recombinant INVS proteins (lanes 1–4) were used for IVK. Akt phosphorylates WT (lane 5), 1–970 (lane 6), and 1–898 (lane 7) INVS, but failed to phosphorylate 1–670 (lane 8) INVS. The arrows (right side) indicated the position of 1-670 fragment (lane 4), which failed phosphorylation (lane 8). The indicated recombinant INVS proteins (lanes 1–10) were used for IVK. Akt phosphorylates WT (lane 6) and 1–898 (lane 7) INVS, but failed to phosphorylate other subfragments of INVS (1–822, 1–746, and 1–670, in lanes 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675–1065 (lane 4), but not 675–822 (lane 5), or 675–746 (lane 6) INVS. Amino acid alignment of 824–898. Motif scan analysis using the Scansite3 database identified three amino acids, T864, S865, and T866, as putative Akt phosphorylation targets. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1–4). In IVK, Akt phosphorylated the WT fragment (824–898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody. Akt phosphorylated WT (lane 9), 1–898 (lane 12) and 675–1065 (lane 15) INVS, but failed to phosphorylate full-length 3A, 675–1065 3A, ∆727–896, 1–857, and 1–822 INVS (lanes 10, 16, 11, 13, and 14, respectively), all of which lacked the T864/S865/T866 target amino acid. Download figure Download PowerPoint Next, we utilized full-length or deletion mutants of INVS for further dissecting the phosphorylation site of INVS by Akt via IVK assays. Akt phosphorylated full-length INVS and 1–898 INVS, but failed to phosphorylate 1–822, 1–746, and 1–670 subfragments of INVS (Fig 2B). We also used additional INVS protein fragments for further dissecting the Akt phosphorylation site(s) on INVS. In contrast to full-length and 1–898 INVS, Akt did not phosphorylate the 1–822, 1–746, and 1–670 subfragments of INVS (Fig 2B). Consistent with these results, Akt phosphorylated a 675–1,065 INVS fragment, but not the 675–822 or 675–746 INVS fragments (Fig 2C). Together, these results indicate that Akt phosphorylates INVS in a region between amino acids 823 and 898. Using the program Scansite (Suizu et al, 2009), we identified three consecutive amino acids (T864, S865, and T866) as putative Akt phosphorylation targets (Fig 2D). These consecutive serine and threonine residues are conserved in mammalian species including Pan troglodytes, Canis lupus familiaris, Bos taurus, or Oryctolagus cuniculus, further supporting the physiological significance of phosphorylation of these residues in vivo (Appendix Fig S1B). Recombinant wild-type (WT) INVS and a triple-alanine-substitution mutant of the three consecutive threonine and serine residues at 864, 865, and 866 (hereafter designated as "3A") within the 824–898 subfragment of INVS were generated. Akt phosphorylated the WT fragment (824–898), but failed to phosphorylate the 3A mutant (Fig 2E). Importantly, Akt phosphorylated WT, 1–898, and 675–1,065 INVS, but failed to phosphorylate 3A mutants in both the full-length and the 675–1,065 subfragment, as well as in the 727–896, 1–857, or 1–822 subfragments, all of which lack the T864/S865/T866 target residues (Fig 2F). Together, these results demonstrate that INVS is a direct substrate of Akt and that Akt phosphorylates INVS at one or several of the serine/threonine residues at T864/S865/T866. INVS and phosphorylated Akt are located at the basal region of the primary cilia PDGF controls the migration, differentiation, and activity of a variety of specialized mesenchymal and migratory cell types, both during development and in the adult animal (Hoch & Soriano, 2003; Schneider et al, 2005, 2010; Christensen et al, 2007). Interestingly, PDGFRα signaling in the primary cilium regulates fibroblast migration via Akt signaling (Schneider et al, 2005; Clement et al, 2013b). Primary cilia organize and extend from the basal body (from the mother centriole) where activated Akt is constitutively present (Zhu et al, 2009), suggesting that Akt may be involved in ciliary development. Using confocal microscopy, we found that active Akt (phospho-S473 Akt) localized to the basal body of primary cilia or to a centrosome-like structure in dividing cells (Fig 3A). Consistent with previous reports, INVS also localized to the proximal region in primary cilia, in the so-called Inversin compartment (Shiba et al, 2009) or ciliary base at the centrosome in dividing cells (Fig 3B). INVS also co-localized with γ-tubulin, a centrosome marker, in confluent cells (Fig 3C). Phosphorylated INVS, detected by an anti-phospho-Akt substrate antibody in immunoblots (see Fig 2), localized to the basal region of the primary cilia in confluent cells (Fig 3D). Upon PDGF-AA stimulation, INVS appeared to be the same position with phospho-Akt substrate at ciliary pocket. PI3K inhibitor (LY294002) inhibited phospho-S473 Akt localization at ciliary pocket (Appendix Fig S2B-E). Figure 3. Both INVS and phosphorylated Akt are located at the basal region of primary cilia Confocal microscopy shows that active Akt (phospho-S473 Akt) is localized in the basal body of primary cilia (arrows) (right-side panels show higher magnification). INVS is localized in the proximal region of the Inversin compartment in primary cilia (arrows) (Inversin compartment, right-side panels show higher magnification). INVS co-localizes with γ-tubulin, a centrosome marker (arrows). Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin staining (right-side panels show higher magnification). Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left-side panels) and INVS (right-side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrows). Schematic representation of the view from an electron microscope. Download figure Download PowerPoint To further dissect the precise localization of Akt and INVS at the ciliary base, we employed a silver-intensified immunogold method for electron microscopy. Our results showed that both phospho-Akt and INVS localized to the basal body, close to the ciliary pocket, only after PDGF-AA treatment (Fig 3E and F). Phosphorylated Akt, but not INVS, is accumulated at the ciliary pocket prior to PDGF-AA treatment (Appendix Fig S2A). PDGF-AA stimulates co-localization of INVS and phosphorylated Akt PDGF-AA is known to mediate intracellular Akt signaling in cilia (Schneider et al, 2005; Christensen et al, 2007; Clement et al, 2013b). Since Akt interacted with and phosphorylated INVS, we next examined whether PDGF-AA stimulation affected the co-localization of INVS and Akt. PDGF-AA stimulation of cells resulted in a time-dependent increase in active Akt–INVS interaction at the basal body (Fig 4A and B). Figure 4. Co-localization of INVS and phosphorylated Akt increases with PDGF-AA treatment PDGF-AA stimulation enhances localization of active Akt (phospho-S473 Akt) at the basal body of primary cilia (arrows) (right-side panels show higher magnification). Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Co-localization was measured by counting yellow pixels (co-localized area) using Imaris software (Bitplane AG). Results presented are means ± SE (n = 31 at time 0, n = 35 at 1 min, and n = 33 at 3 min). Three independent experiments were analyzed with similar results. Statistical significance was determined by Student's t-test. Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT Akt (second panel, lane 1) or phospho-mimetic Akt (T308D/S473D, lane 2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3). PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively). Both WT and 3A-INVS, which are localized at the Inversin compartment of primary cilia under serum-starved condition (left-side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right-side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating co-localization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n = 28). Three independent experiments showed similar results. Statistical significance was determined by Student's t-test. Fluorescence intensities of INVS (green), acetylated α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath. Download figure Download PowerPoint Since Akt phosphorylates INVS, we next examined whether Akt kinase activity affects its interaction with INVS. In co-immunoprecipitation assays, INVS preferentially bound to WT Akt or a phospho-mimetic mutant of Akt (T308D/S473D), but failed to interact with a phosphorylation-deficient Akt mutant (T308A/S473A) (Fig 4C). To further examine the role of Akt activation in Akt–INVS interaction, we stimulated HEK293T cells with PDGF-AA, which mediates Akt signaling in primary cilia. As expected, the interaction of Akt with INVS was clearly augmented by PDGF-AA administration (Fig 4D). Since INVS localizes to the Inversin compartment of primary cilia, we next examined how PDGF-AA affects the localization of INVS (WT vs. 3A). Consistent with a previous report (Shiba et al, 2009), both WT and 3A INVS localized at the Inversin compartment of primary cilia under serum-starved conditions (Fig 4E, left-side panels). After PDGF-AA stimulation, however, INVS relocalized to the proximal portion of primary cilia around the basal body (Fig 4E, right-side panels) where phosphorylated Akt was constitutively present (see Fig 3). Both WT and 3A INVS translocated to the basal body after PDGF stimulation. Akt controls ciliary development through the phosphorylation of INVS at T864/S865/T866 Our observations thus far supported the hypothesis that the interaction between Akt and INVS played an important role in the control of ciliary growth. Therefore, we examined whether Akt phosphorylated INVS at T864/S865/T866 in a physiological context. PDGF-AA treatment, which activates Akt, induced phosphorylation of WT INVS (Fig 5A, top panel). The phosphorylation of INVS could be suppressed by LY294002, MK2206, or GSK690693 inhibitors for Akt, indicating that INVS phosphorylation at T864/S865/T866 was indeed mediated by Akt in a physiological context. An increase in Akt phosphorylation at both S473 and T308 sites was observed after GSK690693 treatment due to a feedback mechanism (Okuzumi et al, 2009). Similar results were obtained after stimulation of HEK293 cells with serum (Appendix Fig S3A) or stimulation of COS-7 cells with PDGF-AA (Appendix Fig S3B). Figure 5. Decrease in ciliary development by phosphorylation-defective INVS at T864/S865/T866 PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively). Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each. Length of primary cilia in confluent hTERT-RPE1 cells was analyzed after siRNA-mediated knockdown of Akt1/2. Ciliary length was decreased in cells expressing siRNA. Results presented are means ± SE of the longitudinal length of acetylated tubulin (red), a marker of primary cilia, in siRNA-transfected (green: EGFP positive) hTERT-RPE1 cells (n = 175 for control siRNA and Akt siRNA, and n = 195 for Akt siRNA plus siRNA-resistant Akt). Three independent experiments showed similar results. Statistical significance was analyzed by Student's t-test. Note that reintroduction of siRNA-resistant Akt (Matsuda-Lennikov et al, 2014) in Akt knockdown cells rescued the effect on ciliary length. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels). Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n = 58 for EGFP vector and for EGFP-INVS WT, and n = 52 for EGFP-INVS 3A mutant). Three independent experiments showed similar results. Statistical significance was determined by Student's t-test. Download figure Download PowerPoint Next, we co-transfected Myr-Akt, a constitutive active form of Akt (Suizu et al, 2009), to induce Akt kinase activity along with WT or 3A INVS to examine the phosphorylation levels of INVS in a physiological context. WT, but not 3A INVS, was phosphorylated by Myr-Akt, de
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