Screening of domain-specific target proteins of polo-like kinase 1: construction and application of centrosome/kinetochore-specific targeting peptide.
Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the Nterminus region, whereas the non-catalytic region in the Cterminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domainspecific target proteins of Plk1, we constructed an Nterminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and Ctermini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.
Ji
Jae-Hoon
JH
Laboratory of Biochemistry, The School of Dentistry, Dankook University, 29 Anseo-Dong, Cheonan-Si, Chungnam 330-714, Korea.
Jang
Young-Joo
YJ
D018797:Cell Cycle Proteins / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008938:Mitosis; D010455:Peptides / Q000008:administration & dosage* / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000097869:Polo-Like Kinase 1