ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice

Abstract

Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking inCaenorhabditis elegans

“…However, whereas in mammalian cells ANKS6 localization to cilia is dependent on the presence of NEK8 (Czarnecki et al 2015), we did not observe changes in the localization of MLT-2 following NEKL-2 depletion (data not shown). In addition, ANKS6/MLT-2 colocalizes with ANKS3/MLT-3 in kidney cells (Delestre et al 2015), and these proteins physically interact through their SAM domains (Leettola et al 2014). However, because MLT-2 does not contain a predicted SAM domain, this interaction may not be conserved in C. elegans, consistent with our observation that MLT-2 and MLT-3 do not extensively colocalize.…”

“…In mammals, mutations affecting NEK8/NEKL-2, ANKS6/MLT-2, INVS/MLT-4, and ANKS3/MLT-3 have been directly linked to a class of diseases termed ciliopathies, which result from defects in the formation of primary cilia Otto et al 2008Otto et al , 2011Trapp et al 2008;Halbritter et al 2013;Hoff et al 2013;Delestre et al 2015). These include kidney disorders, such as nephronophthisis type 2 (Otto et al 2003) and juvenile cystic kidney disease (Liu et al 2002), as well as situs inversus, cardiovascular abnormalities, liver fibrosis, and defects in other organ systems Hoff et al 2013;Manning et al 2013).…”

“…These include kidney disorders, such as nephronophthisis type 2 (Otto et al 2003) and juvenile cystic kidney disease (Liu et al 2002), as well as situs inversus, cardiovascular abnormalities, liver fibrosis, and defects in other organ systems Hoff et al 2013;Manning et al 2013). These findings, as well as the expression of NEK8, INVS, ANKS6, and ANKS3 in the proximal ciliary compartment (Shiba et al 2009(Shiba et al , 2010Hoff et al 2013;Delestre et al 2015), have led to the view that these proteins function primarily to control cilia formation. Our findings, however, suggest that ciliogenesis itself may not be the key central function of these proteins.…”

“…NEKL-2 is orthologous to mammalian NEK8, which has been implicated in ciliogenesis Quarmby and Mahjoub 2005;Otto et al 2008;Shiba et al 2010;Zalli et al 2012). Correspondingly, mutations in NEK8 can lead to organogenesis defects resulting from ciliopathies Otto et al 2008Otto et al , 2011Trapp et al 2008;McCooke et al 2012;Halbritter et al 2013;Hoff et al 2013;Manning et al 2013;Delestre et al 2015). NEKL-3 is the C. elegans ortholog of mammalian paralogs NEK6 and NEK7, which have been reported to control several processes associated with cell division and are linked to human cancer.…”

Metabolic Phenotyping of Anks3 Depletion in mIMCD-3 cells - a Putative Nephronophthisis Candidate

“…Recent findings revealed that a defectively mutated Anks3 is causing laterality defects in an autosomal recessive manner 14 . Furthermore, recent studies revealed Anks6 as a strong interaction partner of Anks3 15 . Interestingly, Anks6 mutations in humans cause NPH with similar phenotypes in animal models 16 – 18 .…”