ABSTRACT: To explore the locations of pseudo-phosphorylated mutant (Plk1(S330/597D);) and non-phosphorylatable mutant (Plk1(S330/597A);) of Plk1 (residues 330 and 597 serine) in HeLa cells and the effect on mitosis.
We transfected wide-type Plk1-GFP, Plk1(S330/597D);-GFP and Plk1(S330/597A);-GFP into HeLa cells, respectively, and detected the locations of wide type and mutants in HeLa cells using immunofluorescence staining and the protein expressions of Plk1(S330/597D);-GFP and Plk1(S330/597A);-GFP with immunoblotting. HeLa cells transfected with Plk1 mutants were observed and analyzed in mitotic phase for cell count and cell cycle by flow cytometry.
The protein expressions of Plk1 mutants were verified. Immunofluorescence microscopy revealed that mutagenesis of these phosphorylation sites did not affect targeting of Plk1 to kinetochore and midbody during mitosis. However, after transfection of Plk1(S330/597A);-GFP, mitosis was inhibited and arrested at cytokinesis compared to control cells. Flow cytometry showed that Plk1(S330/597A);-GFP expression led to an increased G2/M arrest (P<0.01).
Non-phosphorylatable mutant of Plk1 (Plk1(S330/597A);) induces cytokinesis failure and causes the cell cycle arrest at G2/M phase.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2012; 28(10):1016-9.
ABSTRACT: To screen 14-3-3Zeta-interacting proteins by the yeast two-hybrid system and confirm the interaction between 14-3-3Zeta and Polo-like kinase 1 (Plk1).
A bait vector pGBKT7-14-3-3Zeta was constructed to screen the 14-3-3Zeta-interacting proteins from the HeLa cDNA library by yeast two-hybrid system. The interaction between 14-3-3Zeta and Plk1 was further confirmed by immunoprecipitation assay. We also examined the distribution of endogenous 14-3-3Zeta and Plk1 in mitosis using immunofluorescent staining.
Plk1 was identified as a partner of 14-3-3Zeta protein by the yeast two-hybrid system. Immunoprecipitation assay demonstrated the interaction between 14-3-3Zeta and Plk1. Immunofluorescence indicated 14-3-3Zeta was colocalized with Plk1 at the midbody during cytokinesis.
Plk1 is a serine/threonine protein kinase that plays multiple critical roles in centrosome maturation, mitotic chromosome segregation, cytokinesis, and the DNA damage response. The interaction between 14-3-3Zeta and Plk1 suggests 14-3-3 family emerges as a novel player in the mitotic regulation.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 08/2012; 28(8):804-7.
ABSTRACT: Serine/threonine kinases secreted from rhoptry organelles are important virulence factors for Toxoplasma gondii. Among rhoptry proteins, the ROP18 kinase has been identified as a key virulence determinant mediating pathogenesis in T. gondii; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, the interactions between the ROP18 kinase of Toxoplasma gondii and the host cell proteins were analyzed using a yeast two-hybrid technique. The cMyc-ROP18(25-251) fusion proteins expressed by pGBKT7 plasmids in AH109 yeast were bound to host cell proteins from a human fetal brain cDNA library transformed to AH109 yeast using a mating method. Using these selection procedures, we identified seven host proteins that had not previously been reported to interact with ROP18 such as DDB1, TOR1AIP1, integrin, SLC3A2, TPST2, DERL2 and OCIAD1. These host proteins are associated with DNA repair, transcriptional regulation, translation modification, protein degradation and cell adhesion. Our data strongly support the hypothesis that the secreted kinase ROP18 is involved in several complex cellular pathways for the invasion and commandeering of host functions.
Acta tropica 02/2012; 122(3):255-60. · 2.22 Impact Factor