The centrosomal kinase Aurora-A/STK15 interacts with a putative tumor suppressor NM23-H1

Abstract

Alterations in the activity of the centrosomal kinase, Aurora-A/STK15, have been implicated in centrosome amplification, genome instability and cellular transformation. How STK15 participates in all of these processes remains largely mysterious. The activity of STK15 is regulated by phosphorylation and ubiquitin-mediated degradation, and physically interacts with protein phosphatase 1 (PP1) and CDC20. However, the precise roles of these modifications and interactions have yet to be fully appreciated. Here we show that STK15 associates with a putative tumor and metastasis suppressor, NM23-H1. STK15 and NM23 were initially found to interact in yeast in a two-hybrid assay. Association of these proteins in human cells was confirmed by co-immunoprecipitation from cell lysates and biochemical fractionation indicating that STK15 and NM23-H1 are present in a stable, physical complex. Notably, SKT15 and NM23 both localize to centrosomes throughout the cell cycle irrespective of the integrity of the microtubule network in normal human fibroblasts.

Figure 1

STK15 and NM23 interact specifically in the two-hybrid assay. Transformants of S.cerevisiae strain pJ64-4a were grown and re-patched on synthetic complete plates lacking Leu and Trp to select for both pGBT and pGADGH plasmid derivatives. The patches were transferred to Whatman No. 1 filter paper for β-galactosidase assays and the color was developed at 37°C for 1 h. A combination of pGBT8-Ras and pGADGH-Raf served as the positive control.

Figure 2

STK15 and NM23 co-immunoprecipitate. Protein lysates from immortalized IMR90 cells were cleared by centrifugation and immunoprecipitated with anti-STK15 plus STK15 polypeptide antigen competitor (lane 1), anti-STK15 alone (lane 2), normal mouse IgG (lane 3) and anti-NM23-H1 (lane 4). The immunoprecipited proteins were fractionated by SDS–PAGE and transferred to nitrocellulose membrane for western blotting. STK15 and NM23-H1 are indicated by arrows.

Figure 3

STK15 and NM23-H1 co-fractionate. Protein lysates from immortalized IMR90 cells were fractionated on Mono S, Mono Q and Superose 6 columns. Fractions 8–25 of 1 ml volume were collected after the final Superose 6 gel filtration column and constituent proteins were separated by SDS–PAGE for western blotting. STK15 and NM23-H1 are indicated by arrows.

Figure 4

STK15 and NM23-H1 localize to centrosomes throughout the cell cycle. IMR90 cells were plated on glass coverslips and cultured in growth medium for 24 h before fixation with paraformaldehyde. The centrosome was visualized with anti-centrin2 and Texas red-labeled secondary antibodies. (A) STK15 and (B) NM23-H1 were visualized with anti-STK15 and anti-NM23-H1, respectively, and FITC-labeled secondary antibodies. DNA was labeled by Hoechst 33342 or DAPI staining. The composite images are STK15 or NM23 merged with centrin2 and DAPI. The green NM23 or STK15 merged with red centrin2 produces yellow spots in the composite image, indicating that NM23 and STK15 localize to the centrosomes. (C) Cells were labeled with anti-β-tubulin-FITC, anti-NM23-TRITC and anti-STK15-AF647 (Cy5) simultaneously and visualized on the microscope with FITC, TRITC, Cy5 and DAPI filters, respectively. Cell cycle stages are indicated on the left.

Figure 4

STK15 and NM23-H1 localize to centrosomes throughout the cell cycle. IMR90 cells were plated on glass coverslips and cultured in growth medium for 24 h before fixation with paraformaldehyde. The centrosome was visualized with anti-centrin2 and Texas red-labeled secondary antibodies. (A) STK15 and (B) NM23-H1 were visualized with anti-STK15 and anti-NM23-H1, respectively, and FITC-labeled secondary antibodies. DNA was labeled by Hoechst 33342 or DAPI staining. The composite images are STK15 or NM23 merged with centrin2 and DAPI. The green NM23 or STK15 merged with red centrin2 produces yellow spots in the composite image, indicating that NM23 and STK15 localize to the centrosomes. (C) Cells were labeled with anti-β-tubulin-FITC, anti-NM23-TRITC and anti-STK15-AF647 (Cy5) simultaneously and visualized on the microscope with FITC, TRITC, Cy5 and DAPI filters, respectively. Cell cycle stages are indicated on the left.

Figure 4

STK15 and NM23-H1 localize to centrosomes throughout the cell cycle. IMR90 cells were plated on glass coverslips and cultured in growth medium for 24 h before fixation with paraformaldehyde. The centrosome was visualized with anti-centrin2 and Texas red-labeled secondary antibodies. (A) STK15 and (B) NM23-H1 were visualized with anti-STK15 and anti-NM23-H1, respectively, and FITC-labeled secondary antibodies. DNA was labeled by Hoechst 33342 or DAPI staining. The composite images are STK15 or NM23 merged with centrin2 and DAPI. The green NM23 or STK15 merged with red centrin2 produces yellow spots in the composite image, indicating that NM23 and STK15 localize to the centrosomes. (C) Cells were labeled with anti-β-tubulin-FITC, anti-NM23-TRITC and anti-STK15-AF647 (Cy5) simultaneously and visualized on the microscope with FITC, TRITC, Cy5 and DAPI filters, respectively. Cell cycle stages are indicated on the left.

Figure 5

(Previous two pages and above) The centrosome localization of STK15 and NM23-H1 is not microtubule dependent. (A) IMR90 cells grown on coverslips were treated with or without nocodazole and fixed for immunofluorescence. The centrosome was visualized by staining with anti-centrin2 and anti-γ-tubulin antibodies. As predicted, the signal representing centrosome-associated γ-tubulin becomes significantly weakened, especially in mitotic cells (prometaphase cells here), upon nocodazole treatment (labeled w/ noc on the left), as compared to that seen in cells without nocodazole (labeled w/o noc on the left). (B) STK15 and (C) NM23-H1 still localize to centrosome in nocodazole-treated cells throughout the cell cycle. IMR90 cells treated with or without nocodazole (w/o noc) were fixed and triple labeled for β-tubulin (FITC), STK15/NM23-H1 (Cy5), centrin2 (Texas red) and DNA (DAPI). The nocodazole treatment totally dissipates the microtubule spindles so the cells with nocodazole fail to show the characteristic β-tubulin staining pattern (the β-tubulin columns). Cell cycle stages are indicated on the left.

Figure 5

(Previous two pages and above) The centrosome localization of STK15 and NM23-H1 is not microtubule dependent. (A) IMR90 cells grown on coverslips were treated with or without nocodazole and fixed for immunofluorescence. The centrosome was visualized by staining with anti-centrin2 and anti-γ-tubulin antibodies. As predicted, the signal representing centrosome-associated γ-tubulin becomes significantly weakened, especially in mitotic cells (prometaphase cells here), upon nocodazole treatment (labeled w/ noc on the left), as compared to that seen in cells without nocodazole (labeled w/o noc on the left). (B) STK15 and (C) NM23-H1 still localize to centrosome in nocodazole-treated cells throughout the cell cycle. IMR90 cells treated with or without nocodazole (w/o noc) were fixed and triple labeled for β-tubulin (FITC), STK15/NM23-H1 (Cy5), centrin2 (Texas red) and DNA (DAPI). The nocodazole treatment totally dissipates the microtubule spindles so the cells with nocodazole fail to show the characteristic β-tubulin staining pattern (the β-tubulin columns). Cell cycle stages are indicated on the left.

Figure 5

(Previous two pages and above) The centrosome localization of STK15 and NM23-H1 is not microtubule dependent. (A) IMR90 cells grown on coverslips were treated with or without nocodazole and fixed for immunofluorescence. The centrosome was visualized by staining with anti-centrin2 and anti-γ-tubulin antibodies. As predicted, the signal representing centrosome-associated γ-tubulin becomes significantly weakened, especially in mitotic cells (prometaphase cells here), upon nocodazole treatment (labeled w/ noc on the left), as compared to that seen in cells without nocodazole (labeled w/o noc on the left). (B) STK15 and (C) NM23-H1 still localize to centrosome in nocodazole-treated cells throughout the cell cycle. IMR90 cells treated with or without nocodazole (w/o noc) were fixed and triple labeled for β-tubulin (FITC), STK15/NM23-H1 (Cy5), centrin2 (Texas red) and DNA (DAPI). The nocodazole treatment totally dissipates the microtubule spindles so the cells with nocodazole fail to show the characteristic β-tubulin staining pattern (the β-tubulin columns). Cell cycle stages are indicated on the left.