Fig 1.
Identification of a CDK5RAP2-interacting protein, Cep169/ NCKAP5L.
(A) CDK5RAP2, a human homolog of CNN, has two conserved motifs, CM1 (red) and CM2 (blue). The N-terminal region of Cep169/NCKAP5L interacts with the CDK5RAP2 CM1 motif by yeast two-hybrid assay and immunoprecipitation (IP) experiments. The amino acid sequence of the N-terminal region is conserved in vertebrates. (B) Interaction of the CM1 motif in CDK5RAP2 with the N-terminal region of Cep169/NCKAP5L, as demonstrated by yeast two-hybrid assay. (C) FLAG-Cep169/NCKAP5L interacts with CDK5RAP2 in the doxycycline-inducible FLAG-NCKAP5L HeLa cell line. Complex formation was detected by immunoprecipitation with mouse anti-FLAG antibody, followed by Immunoblotting (IB) with anti-CDK5RAP2 antibody. (D) Cep169/NCKAP5L interacts with CDK5RAP2 via its N-terminal region. 293T cells were co-transfected with GFP-Cep169/NCKAP5L wild type or its various deletion mutants together with FLAG-CDK5RAP2. (E) Sequence alignment of Cep169/NCKAP5L interaction domain with CDK5RAP2 from different species are as follows: human, mouse, rat, dog, cow, and frog. GenBank accession number of human Cep169/NCKAP5L: NC_000012.11.
Fig 2.
Cep169/NCKAP5L is a centrosomal protein with molecular weight of 169 kDa.
(A) IB of whole-cell lysates from HeLa, U2OS, and 293T cells treated with control Luciferase (Luc) siRNA or Cep169/NCKAP5L siRNA, and detected with anti-Cep169 and anti-β-tubulin antibodies. (B) IB of whole-cell lysates transfected with control (Luc siRNA) or two independent siRNA oligonucleotides (Cep169-1 and Cep169-2 siRNAs), probed with antibodies against Cep169 and β-tubulin. (C) Determination of polypeptide molecular size for human Cep169/NCKAP5L by silver staining. The silver staining bands of FLAG-Cep169 observed in the presence of doxycycline disappeared at right lane in the absence of doxycycline. Graph showing estimation of polypeptide size (molecular weight 169 kDa) for Cep169/NCKAP5L. (D) Immunofluorescence images of U2OS cells probed with anti-Cep169 and γ-tubulin antibodies (left). (Scale bar, 10 μm.) U2OS cells were treated with control (Luc siRNA) or Cep169/NCKAP5L siRNA. The centrosomal intensity of γ-tubulin in the U2OS cells (right). (n = 30 cells for each case; error bars, S.E.). (E) Immunofluorescence images of FLAG-Cep169-expressing mitotic HeLa cells probed with anti-FLAG and γ-tubulin antibody. (Scale bar, 10 μm.).
Fig 3.
Cep169 targets MT distal ends in a manner dependent on the interaction with EB1.
(A) Immunofluorescence images showing co-localization of FLAG-tagged CDK5RAP2 and GFP-Cep169 at centrosome (arrowheads) and distal ends in U2OS cells probed with anti-FLAG antibody. (Scale bar, 10 μm). (B) Immunofluorescence images showing co-localization of FLAG-Cep169 and EB1 at distal ends in stable U2OS cell probed with anti-FLAG and EB1 antibodies. (Scale bar, 10 μm). (C) Schema of Cep169 showing the three (S/T)x(I/L)P motifs (up) and their alanine-substituted mutants (bottom). (D) Immunofluorescence image of Cep169 localization in U2OS cells transfected with FLAG-tagged wild type or alanine-substituted mutants of each (S/T)x(I/L)P motif in Cep169, as described in (C). (Scale bars, 10 μm). (E) Relative intensity of wild type or mutant Cep169 at distal ends. (n = 60 cells for each case; error bars, S.E.). P values result from t-test (***P < 0.0001, NS: not significant). (F) Pull-down assays using recombinant GST-EB1 as an affinity matrix to isolate FLAG-Cep169 WT or SxAA123 mutant. FLAG-Cep169 or GST and GST-EB1 proteins were analyzed with IB using anti-FLAG antibody or Coomassie Brilliant Blue (CBB) staining, respectively.
Fig 4.
(A) U2OS cells were transiently transfected with GFP-Cep169. The cells were stained for acetylated MTs (anti-acetylated α-tubulin antibody). (Scale bar, 10 μm). (B) U2OS cells stably expressing EB1-GFP were transiently transfected with mRFP-Cep169 and FLAG-CDK5RAP2. The cells were stained for anti-FLAG antibody. (Scale bar, 10 μm). (C) CDK5RAP2, but not Cep169, has MT organizing center (MTOC) activity. U2OS cells were transfected with FLAG-CDK5RAP2 or FLAG-Cep169 and immunostained with anti-FLAG and anti-γ-tubulin antibodies. Expression of CDK5RAP2, but not Cep169, caused formation of cytoplasmic aggregates (arrowheads) associated with γ-tubulin. (Scale bars, 10 μm). (D) Immunofluorescence images of cells probed with anti-β-tubulin antibody (red) and DAPI (blue) in control Luc siRNA cells or Cep169 siRNA cells and then fixed after cold treatment for 15 min. (Scale bars, 20 μm).