The centrosomal kinase Plk1 localizes to the transition zone of primary cilia and induces phosphorylation of nephrocystin-1

Abstract

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.

Figure 1. Plk1 colocalizes with NPHP1 at the base of the cilium.

A Ciliated hTERT-RPE1 (human retinal pigmented epithelial cells and HK2 human kidney cells were stained with antibody to Plk1 (green), acetylated α-tubulin (orange), and γ-tubulin (red), and treated with DAPI to visualize DNA (blue). The scale bar represents 5 µm. B Ciliated hTERT-RPE1 cells and HK2 cells were stained with antibody to acetylated α-tubulin (orange), γ-tubulin (red), to NPHP1 or Plk1 as indicated (green), and with DAPI to visualize DNA (blue). The third row shows merged signals from staining with antibody to Plk1 (orange), NPHP1 (green) acetylated α-tubulin (orange), γ-tubulin (red) and DAPI was used to visualize DNA (blue). The scale bar represents 5 µm.

Figure 2. Plk1 associates with NPHP1.

A Western blot of immunoprecipitates (IP) or lysates (Lys) from HEK293T cells co-transfected with plasmids expressing V5-tagged NPHP1 and Flag-tagged Plk1 or negative control protein (Eps1–225 [13]). β-actin was assessed as a loading control. B Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 or empty Flag vector. C Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells transfected with plasmid expressing Flag-tagged NPHP1 or the negative control protein (Eps1–225 [13]). Endogenous Plk1 was detected using a specific antibody against Plk1. D A panel of Flag-tagged NPHP1 derivatives, including truncations, internal deletions and a T87A mutant, was analyzed by co-immunoprecipitation with Myc-tagged Plk1. E Western analysis of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 constructs as indicated, or the Flag-tagged control protein (Eps1–225). * indicates immunoglobulin heavy chain.

Figure 3. Plk1 directly phosphorylates NPHP1 in vitro.

A Alignment of NPHP1 protein sequences from multiple species indicates a conserved candidate Plk1 motif at position T87. B An in vitro kinase assay performed with active Plk1 and recombinant His-fused NPHP1 protein indicates phosphorylation within the NPHP1 N-terminal 205 amino acids. CB, Coomassie Blue.

Figure 4. Plk1 is activated during ciliary disassembly but has limited influence on disassembly dynamics.

A Immuno blot analysis of whole cell lysates prepared from hTERT-RPE1 grown under starved, ciliated conditions (0h) or at the times indicated following serum treatment to induce ciliary resorption. Ph-Plk1^T210 represents activated Plk1; B, C Quantification of the expression of (B) Ph-Plk1^T210 or (C) total Plk1 normalized to β-actin and relativized to the expression level without serum induction. Results from three independent experiments were calculated. * P<0.05, ***P≤0.005. Error bars represent SE. D Upper panel: Immunofluorescence of starved ciliated hTERT-RPE1 cells treated with the Plk1-inhibitor BI 2536 or with DMSO for 3 hours, then maintained in serum-free medium, or induced for ciliary disassembly with 10% serum-containing medium. Lower panel: Immunofluorescence of serum-starved, ciliated hTERT-RPE1 cells treated with siRNA to Plk1 or with scrambled (scr) control siRNA after 0, 2 and 24 hours of serum addition. Image shows acetylated α-tubulin (green), γ-tubulin (red) and DNA (blue). The scale bar represents 10 µm. E - H Quantification of three independent repetitions of experiment presented in D. E and G Quantification of the percentage of ciliated cells, with an average of 250 cells counted for each condition. F and H Quantification of cilia length, with an average of 50 cilia measured for each condition. * P<0.05, **P<0.01. ***P<0.001. Error bars represent SE.