Abnormal AurA expression and activity in renal cysts. (a) Western blot analysis of total AurA levels in human cells: HEK293, human embryonic kidney cells; HK-2, adult human kidney epithelial cells; RPE1, immortalized retinal pigment epithelial cells; HOSE, primary human ovarian surface epithelial cells; MCF10A, immortalized mammary epithelial cells. For all experiments, β-actin was used as a loading control. (b) Immunofluorescence of HK-2 cells treated with siRNA to deplete AurA (siAurA) or with control scrambled siRNA (Scr). Bar, 10 µm. (c) Immunohistochemical detection of AurA and phAurA in renal cysts from PKD patients. Epithelial cells lining cysts and cyst-associated areas of hyperproliferative epithelial cells, but not cyst-associated fibrotic tissue, consistently demonstrate strong staining. Molecular masses are given in kilodaltons.

PHA-680632 enhances PC2 activity at low IC values. (a) IC₅₀ curve for PHA-680632 (PHA) treatment of HK-2 cells stably expressing PC2. Cell viability was detected by Alamar blue assay 72 h after treatment. (b) HK-2 cells stably expressing PC2 were treated with DMSO or with PHA-680632 at concentrations of 0.5 or 3.25 µM for 2 or 24 h before AVP stimulation and determination of the F/F₀ ratio. Addition of AVP is indicated by the first arrow. (c) Quantification of relative F/F₀ amplitude of data in b. Difference between the double asterisks and single asterisks is significant, P = 0.0022; the difference between the single asterisks is not significant. Analysis as AUC is shown in Fig. S3 d. F, fluorescence intensity; F₀, baseline fluorescence intensity. Error bars indicate SEM.

AurA phosphorylates PC2 on C-terminal residue S829. (a) Structural motifs on PC2 C-terminal (CT) domain include PC1-binding motif (coiled-coil domain; aa 832–895), an EF hand for Ca²⁺ binding (EF-hand; aa 754–782), and ER-targeting sequences (aa 787–820). Location of assessed sites for CK2 (S812) and AurA (S829 and S944) phosphorylation are indicated. NT, N-terminal. (b) Bacterially expressed GST-PC2_779–968 or GST-NEDD9_1–363 and MBP (positive controls) or histone H1 (H1) were incubated with recombinant active AurA kinase in an in vitro kinase assay with γ-[³²P]ATP. The reactions were resolved by SDS-PAGE and analyzed by autoradiography, Coomassie blue (CB) staining, and Western blotting with the antibody to AurA. (c) Experiment as in b, except GST-NEDD9_1–363 was included in the indicated reactions, and relative phosphorylation of PC2 and MBP were calculated (graph). Data are expressed as mean values ± SEM of three experiments. *, P < 0.001. For analysis, phosphorylation of PC2 and MPB was first normalized to total PC2 and total MBP and then further normalized to AurA levels. The boxes indicate phosphorylated species of the C-terminal tail of PC2. (d) Bacterially expressed GST-PC2_779–968 was incubated with recombinant preactivated AurA (AurA active) or AurA purified from baculovirus in an in vitro kinase assay in the presence or absence of PC2, CaM, and Ca²⁺ as indicated. (top) autoradiography; (bottom) Western blots with the indicated antibodies. (e and f) GST-fused derivatives of PC2_779–968 with mutations affecting a previously defined CK2 site (PC2_S812A) and two candidate AurA sites (PC2_S829A and PC2_S944A) alone or in combination were used in in vitro kinase reactions with recombinant AurA. Phosphoimaging of autoradiographs from three independent experiments was quantified (f), and the relative phosphorylation of PC2_779–968 mutants was calculated (graph). Data are expressed as mean values ± SEM of three experiments. *, P < 0.001. (g and h) Experiments as in e and f, except recombinant CK2 was used rather than AurA. *, P < 0.001. (i) Lysates from HEK293 cells overexpressing Myc-tagged PC2 or the Myc-tagged S829A PC2 mutant (Myc) in the presence or absence of AurA or T288D (catalytically active) AurA were used for immunoprecipitation. Cells were treated with 10 µM H89 PKA inhibitor or 500 nM PHA-680632 (PHA) AurA inhibitor for 3 h as indicated. RRxS-directed antibody was used to visualize phosphorylation of the S829 residue (phPC2). Western blot of coimmunoprecipitation from three independent experiments was quantified (j), and relative phosphorylation of PC2 was calculated. Difference versus PC2: *, P < 0.05. WT, wild type.

Abundant AurA expression and activity in normal kidney tissue and cells. (a and b). Immunohistochemical visualization of endogenous total AurA (a) or phAurA (b) in normal human kidney tissue. Typical fields from the cortical (left) and medullary regions (center) are shown. PCT, proximal convoluted tubules; DCT, distal convoluted tubules; CD, collecting ducts; LH, thin segments of Loops of Henle; GM, glomerulus. (c and d) Immunohistochemical visualization of endogenous AurA (c) or phAurA (d) in normal mouse kidney tissue. (right) Sections stained with antibody to AurA preincubated with blocking peptide and, for phAurA, negative control IgG.

AurA negatively regulates basal cellular Ca²⁺ levels and PC2-dependent Ca²⁺ channel activity. (a) Effect of PHA-680632 on basal intracellular Ca²⁺ in HK-2 and LLCPK1 cells. Cells were loaded with 5 µM Fluo-4 AM, which autofluoresces upon binding cytoplasmic Ca²⁺. Cells were treated with PHA-680632 (PHA) to inhibit Aurora kinase, or DMSO vehicle control. *, P = 0.0024; **, P = 0.0013. (b) Basal Ca²⁺ level in LLCPK1 cells treated with siRNA to deplete AurA (siAurA) versus scrambled siRNA control (Scr). *, P = 0.00056. (c) Basal Ca²⁺ levels were assessed in Pkd2^−/− and Pkd2^+/− cells treated with PHA-680632 or DMSO. *, P = 0.0132; **, P = 0.0209 (in comparison with DMSO-treated Pkd2^+/− cells); and P > 0.05 (NS) in comparison with DMSO-treated Pkd2^−/− cells. (d) Basal Ca²⁺ levels were assessed in two PKD1 mutant cell lines, WT9-7 and WT9-12, treated with PHA-680632 or DMSO. *, P = 0.0098; **, P = 0.0107. For a–d, all data are expressed as the means ± SEM from at least three independent experiments (n > 50 in each experiment). (e) HEK293 cells transiently cotransfected 48 h previously with plasmids expressing PC2 and AurA-RFP or control RFP were loaded with 5 µM Fluo-4 AM before analysis. Immunofluorescence was measured before and after addition of AVP (indicated by arrows in e, g, and i). Data are presented as the F/F₀ ratio, indicating increase in signal over unstimulated cells. F, fluorescence intensity; F₀, baseline fluorescence intensity. (f) For this and subsequent experiments, the mean increase in amplitude (±SEM) of AVP-induced cytoplasmic Ca²⁺ transients was calculated from n > 50 RFP-positive cells in each of three experiments. The asterisks shows that, for AurA-RFP, amplitude decreased versus RFP controls; P = 0.008. Alternative analysis as AUC is shown in Fig. S3 a. (g) HEK293 cells transfected 48 h previously with a plasmid expressing PC2 or vector control and pretreated for 2 h with 500 nM PHA-680632 or DMSO vehicle were analyzed as in e. (h) Quantification of data in f. The asterisk indicates that PHA-680632 increased amplitude of signal versus DMSO; P = 0.0007. Analysis as AUC is shown in Fig. S3 b. (i) siRNA depletion of AurA in HK-2 cells increases amplitude of Ca²⁺ release versus scrambled control. (j) Quantification shows significant difference in amplitude (*, P = 0.0055). Analysis as AUC is shown in Fig. S3 c.

AurA interacts directly with PC2. (a) Antibody to AurA or control IgM was used for immunoprecipitation (IP) from HK-2 cells (left), mouse kidney lysates (middle), or PKD2^−/− and PKD2^+/− cells (right) followed by Western blotting with antibodies to AurA or PC2 (YCC2) as indicated. The strong band migrating at ∼110 kD represents native PC2, with faster migrating bands reflecting degradation products and slower migrating bands showing glycosylated species. (b) Antibody to AurA was used for immunoprecipitation from whole-cell lysates (WCL) transfected with plasmids expressing AurA and either GFP-PC2_779–968 or GFP followed by Western blotting with the indicated antibodies. Reverse orientation immunoprecipitation is shown in Fig. S2 f. (c) Antibody to AurA was used in immunoprecipitation from the in vitro mixture containing GST-PC2_779–968 or GST only and recombinant His-AurA. CB indicates Coomassie blue staining of starting material. Reverse orientation pull-down is shown in Fig. S2 g. (d) RFP-fused catalytic (cat; aa 132–403) and noncatalytic (noncat; aa 1–131) domains of AurA or full-length (FL) AurA-RFP coexpressed with Flag-PC2_779–968 into HEK293 cells and immunoprecipitated with the Flag antibody followed by Western blotting with the indicated antibodies. (e) HEK293 cells were cotransfected with plasmids expressing combinations of GFP, GFP-PC2_779–968, or Flag-PC1-CT_4191–4302 and AurA as indicated. Cell lysates were immunoprecipitated using the anti-GFP antibody and visualized with antibodies indicated in the Western blot analysis. (f) Plasmids expressing HA-NEDD9, HA-BioB, and GFP-PC2_779–968 were transfected into HEK293 cells, cell lysates were immunoprecipitated with the antibody to GFP, and Western blotting was performed with the indicated antibodies. The asterisk indicates immunoglobulin heavy chain. Molecular masses are given in kilodaltons.