Polo-box motif targets a centrosome regulator, RanGTPase.
Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk1 localizes in the centrosome, the central spindle, the midbody as well as the kinetochore. The non-catalytic region in the C-terminus of Plk1 has conserved sequence motifs, named polo-boxes. These motifs are important for Plk localization. GFP protein fused with the core sequences of polo-box (50 amino acids) localized Plk to target organelles. We screened for Plk interacting proteins by constructing a tandem repeat of the polo-box motif, and used it as bait in the two-hybrid system with HeLa cell cDNA library. RanGTPase was detected as a positive clone. Through in vitro and in vivo protein binding analysis in synchronized cells by thymidine block and by nocodazole treatment, we confirmed the interaction between endogenous Ran and Plk1. We showed that endogenous Ran and Plk1 proteins were co-localized to centrosomes, which is a major target organelle of endogenous Plk1, in early mitotic cells by immunofluorescence. Finally, we demonstrated that Plk1 phosphorylated RanBPM, a Ran-binding protein in microtubule organizing center, through the interaction with Ran. These data suggested that the core motif of polo-box is sufficient for Plk1-targeting, and that Plk1 may play roles in centrosome through recruitment and/or activation of Ran/RanBPM proteins.
Jang
Young-Joo
YJ
Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oeun-Dong, Yusong-Gu, Daejeon 305-333, Republic of Korea. youngjoo@kribb.re.kr
Ji
Jae-Hoon
JH
Ahn
Ji-Hee
JH
Hoe
Kwang-Lae
KL
Won
Misun
M
Im
Dong-Soo
DS
Chae
Suhn-Kee
SK
Song
Sukgil
S
Yoo
Hyang-Sook
HS
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D018797:Cell Cycle Proteins; D018385:Centrosome / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000097869:Polo-Like Kinase 1
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D020931:ran GTP-Binding Protein; D000097869:Polo-Like Kinase 1; C116005:Ran binding protein 9