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

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.

Perspective: Potential Impact and Therapeutic Implications of Oncogenic PI3K Activation on Chromosomal Instability

“…Data are from cBioportal [144,145] (accessed June 2019). [146,147] p85 can associate with the centrosome in an insulin-dependent manner [147] Akt/PKB [108] Akt/PKB is phosphorylated during mitosis and is present in the centrosome [148] T308-phosphorylated Akt/PKB is present in basal body of primary cilia [149] S473-phosphorylated Akt/PKB is present in basal body of primary cilia. Akt/PKB interacts with and phosphorylates the ciliary protein Inversin.…”

“…[152] Phosphatase-independent (scaffold function) of PTEN: is recruited to premitotic centrosomes in a Plk1-dependent fashion [151] TSC1 present in centrosomephosphorylated TSC1 and phosphorylated TSC2 co-immunoprecipitate with Plk1 GSK3β [108] Phospho-GSK3 at the centrosomes upon entry into mitosis Inversin [149] Akt/PKB interacts with and phosphorylates the ciliary protein Inversin -dimerization. Co-localization of Inversin and phosphorylated-Akt/PKB at the basal body is augmented by PDGF-AA.…”

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

“…Approximately 10 independent contacts between peroxisome and primary cilium occurred in 2 h, and the contacts continued for 10-20 min in the untreated hTERT-RPE1 cells containing approximately 150-200 peroxisomes labeled with ECFP-SKL (Appendix Fig S4, Movie EV1). To precisely determine the spatial interaction between peroxisomes and primary cilia, we stained endogenous peroxisomes and primary cilia with anti-PMP70 (peroxisomes), antininein (basal bodies), anti-ARL13B (ciliary axonemes), and antiphospho Ser473 Akt (ciliary pockets) antibodies (Suizu et al, 2016). Some PMP70-positive spots partially overlapped with the phospho Ser473-Akt spot at the ciliary pocket in around 15% of the hTERT-RPE1 cell population (Fig 3B and C).…”

The nucleoside-diphosphate kinase NME3 associates with nephronophthisis proteins and is required for ciliary function during renal development

“…However, the localization of NPHPs into distinct subciliary zones is not mutually exclusive. For example, NEK8 and INVS have also been observed both in the proximal axoneme and at the basal body ( 5 , 14 , 15 ).…”