ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning

Abstract

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.

Figure 1. ANKS6 interacts with NEK8, enhancing NEK8 kinase activity

a – IP of FLAG-NEK8 from mIMCD3 cells stably transduced with a retroviral FLAG-NEK8 expression construct reveals a number of distinct coprecipitating bands on silver stain. ANKS6 is identified by mass spectrometry. b – Mass spectrometric analysis of the FLAG-IP eluate reveals a number of distinct peptide fingerprints, amongst which ANKS6 was elected as an interesting candidate protein for further analysis. c – ANKS6 co-precipitates with NEK8, is phosphorylated by NEK8 and promotes NEK8 autophosphorylation in an IP/kinase assay. No phosphorylation activity was observed in the absence of ANKS6, or in the setting of a K33M “kinase-dead” allele. d – ANKS6 enhances kinase activity of wildtype NEK8, as well as the pathogenic NEK8^jck mutant, towards itself, the kinase, and towards α-casein, a nonspecific phosphorylation substrate.

Figure 2. ANKS6 exerts its kinase stimulating activity through direct interaction with the NEK8 kinase domain

a – NEK8 truncations were employed in IP/binding assays in order to map interaction sites. b – The kinase domain of NEK8, along with a short C-terminal extension (NEK8^1–295) mediates the interaction with ANKS6, while the RCC1-domain does not contribute to complex formation. c – NEK8 interacts with inversin through its RCC1-domain; the five C-terminal RCC1-repeats (amino acids 416–698), in particular, are necessary and sufficient for binding. d – The NEK8 kinase domain (NEK8^1–258), as predicted by homology alignment, is not capable of binding ANKS6 and is inactive as a kinase. NEK8^1–295, a larger truncation that binds ANKS6 exhibits dramatically enhanced phosphorylation activity. The full-length wildtype NEK8 and the NEK8¹⁴¹⁵ truncation are included as controls. Any phosphorylation is only detected in the presence of ANKS6, generating autoradiographic signals at the levels of ANKS6 (100 kDa), α-casein (25 kDa) and the molecular weight of the respective NEK8 truncations. e – Co-IP of myc-ANKS6 with FLAG-NEK8, INVS and -NPHP3, and fractionation with different ionic strength conditions demonstrates formation of a salt-insensitive, robust subcomplex between NEK8 and ANKS6.

Figure 3. The ankyrin-repeat domain of ANKS6 is necessary for NEK8 binding and targeting of ANKS6 to the inversin compartment

a – Different ANKS6 truncations were employed in IP/binding assays and IF. b – ANKS6 interacts with NEK8 through its ANK-repeat domain, while the SAM-domain is not essential for binding. ANKS6 truncations that interact with NEK8 are readily phosphorylated in complex with NEK8. c – A truncation variant of ANKS6 lacking the first ANK-repeat binds to NEK8, while truncation of the first four ANK-repeats completely abrogates binding. d, e – shRNA-mediated targeted knockdown of NEK8 significantly reduces the ciliary ANKS6-signal. IF signal intensities were compared between wildtype IMCD cells and IMCD^shNek8 cells, for both anti-NEK8 and anti-ANKS6 signals (scale bar: 2 µm). The number of individual cilia analyzed is indicated in the respective bar. Differences were highly significant by Student’s t-test with p<0.01 (upper bar graph). The total level of ciliation is only mildly affected by Nek8 knockdown (lower bar graph; percentage of numeric counts of ciliated cells over total cells). f – Fluorescence immunohistochemistry on kidney sections of wildtype (upper panels) and Nek8^−/− mice (lower panels), employing antibodies against ANKS6 (green) and acetylated tubulin (red). Positive ANKS6 signals at the ciliary base can only be identified in the wildtype, not in Nek8^−/− cilia (scale bar: 5 µm). g, h – Coexpression of myc-tagged ANKS6 truncation variants and FLAG-tagged wildtype NEK8 demonstrates the importance of the ANK-repeat domain for NEK8-binding and consecutive ciliary targeting of ANKS6 (scale bar: 2 µm; upper bar graph: percentage of numeric counts of ANKS6-positive cilia over total cilia; lower bar graph: percentual level of ciliation; the number of cells counted is indicated in each bar).

Figure 4. Homozygous Anks6^Strkr and Nek8^Roc mutants exhibit a ciliopathy syndrome

a–i – Homozygous Anks6^Strkr mutants exhibit a spectrum of left-right patterning defects and complex CHD. a – Situs solitus, b – situs inversus and c – situs ambiguus or heterotaxy presentations in homozygous Anks6^Strkr mutant mice. Note the presence of dextrocardia with right-sided stomach in the heterotaxy mutant. d – In a heterotaxy mutant, the aorta is transposed anterior to the pulmonary artery, yielding TGA. Also, note duplication of the inferior vena cava (IVC). e – Heterotaxy mutants typically exhibit right pulmonary isomerism with four lung lobes bilaterally. f–i – Histology and 3D reconstruction using ECM allowed detailed interrogation of intracardiac anatomy for CHD diagnosis. Shown are Anks6^Strkr mutants exhibiting TGA with aorta inserted into the RV (f); atrioventricular septal defects (AVSD) (g); right atrial isomerism with symmetrical insertion of the superior vena cava (RSVC, LSVC in h) into the left and right atria, respectively, and abnormal duplication of the IVC (i), and right pulmonary isomerism with four lung lobes bilaterally (i). j–o – Homozygous Nek8^Roc mutants exhibit a spectrum of left-right patterning defects including situs inversus (j) and heterotaxy (k). l – A mutant is shown exhibiting thoracic situs with left pulmonary isomerism, as indicated by only one right and left lung lobe. m – Duplicated IVC can also be observed. n, o – Histological analysis with ECM showed double outflow right ventricle with positioning of both the aorta (Ao) and pulmonary artery (PA) over the RV (n) and a large ventricular septal defect (VSD). Also observed is an atrioventricular septal defect (AVSD in panel o). Black scale bars: 200 µm; white scale bars: 500 µm.

Figure 5. Cystic kidney phenotypes in Anks6^Strkr and Nek8^Roc mutants

a–f – Hematoxylin and eosin stained kidney sections of newborn wildtype (a, d), homozygous Anks6^Strker (b, e) and Nek8^Roc mutant mice (c, f). In the Anks6 mutant kidney, note the presence of widespread cystic tubular dilatation throughout the cortex and medulla, along with a number of markedly enlarged glomerular cysts. The Nek8^Roc mutants showed hydronephrosis with hydroureter and cystic tubular dilatation throughout the parenchyma along with some small glomerular cysts (scale bar: 100 µm). g–i – T1α (red; glomeruli), DBA (green; collecting ducts) and DAPI (blue; nuclei) staining in these same kidneys showed cystic dilatation of Bowman’s space in the glomerulus in the Anks6 (h) and Nek8 (i) mutant (scale bar: 10 µm). j–l – Acetylated tubulin (red; cilia), DBA (green; collecting ducts) and DAPI (blue; nuclei) staining shows the presence of shortened cilia in the mutant Anks6^Strkr and Nek8^Roc newborn kidneys (scale bar: 10 µm). m–p – Hematoxylin and eosin stained kidney sections of adult wildtype (m, o) and homozygous Anks6^Strkr (n, p) adult kidneys showed large glomerular cysts throughout the cortex and cystic tubular dilatation along the corticomedullary junction of the kidney along with inflammatory mononuclear cell infiltrates in the mutant (n). Scale bar: 100 µm. q, r – T1α (red; glomeruli), DBA (green; collecting ducts) and DAPI (blue; nuclei) staining of adult kidneys showed cystic dilatation of Bowman’s space in the glomerulus in the Anks6 mutant (r; scale bar: 10 µm). s, t – Acetylated tubulin (red; cilia), DBA (green; collecting ducts) and DAPI (blue; nuclei) staining of adult kidneys showed cystic dilatation of the collecting ducts and nephrons in the Anks6 mutant (t). Note that tubular epithelia are ciliated in both control and mutant (scale bar: 5 µm).

Figure 6. Anks6^M187K and Nek8^I124T mutations exert their pathogenicity through a functional inactivation of the NEK8 kinase

a, b – IF analysis of FLAG-tagged NEK8 with myc-tagged wildtype and ANKS6^M187K mutant proteins suggests that mutant protein is capable of localizing to the IC (scale bar: 2 µm). c – Fluorescence immunohistochemistry of kidney sections of wildtype (left), Anks6^Strkr mice (middle) and Nek8^Roc mice (right), employing antibodies against native ANKS6 protein (green) and acetylated tubulin (red; scale bar: 5 µm). ANKS6 positivity at the base of renal tubular epithelial cilia is noted in all examined genotypes. d – Co-IP of myc-tagged wildtype ANKS6 (positive control), the ΔANK-repeat domain (negative control) and the ANKS6^M187K mutant with FLAG-Nek8 reveals a markedly reduced binding affinity of the mutant, e – Co-IP of wildtype and mutant ANKS6 with NEK8 at different NaCl concentrations. The interaction of NEK8 with the ANKS6^M187K mutant occurs at a lower affinity than with the wildtype, but is also independent of ionic strength. f – Co-IP and kinase assays of NEK8 in presence or absence of ANKS6 and the ANKS6^M187K mutation. The coprecipitated ANKS6^M187K mutant protein fails to sufficiently stimulate NEK8 phosphorylation activity. g – All examined NEK8 alleles (wildtype, K33M, I124T) are capable of translocating to the IC and to recruit ANKS6, as shown by IF of FLAG-NEK8 transfected IMCD-3 shNek8 cells (anti-FLAG, green), and counterstain for endogenous ANKS6 (red; scale bar: 2 µm). h – Co-IP and kinase assays reveal that NEK8^I124T interaction with ANKS6 is unimpaired, as compared with the wildtype. However, the NEK8^I124T/ANKS6-complex is functionally “kinase-dead” and reveals a negligible extent of phosphorylation activity, as compared with ANKS6-associated wildtype NEK8 (positive control) and NEK8^K33M (negative control).