Isolation of Centrosome-Associated Polo-Box Domain-Binding Proteins

1. Make Primers Cloning procedures GST Polo-Box Domain Pull down assay Western/Immuno Blotting P p-T78 peptide : PLHST78AI(in vitro by Plk) Nhe1(dead) AscI HA tag PmeI EcoRV XhoI NotI 491 WT, AM, Input 485 WT, AM, Input 497 WT, AM, Input 521 WT, AM, Input 509 WT, AM, Input 517 WT, AM, Input Plk1 Mitotic entry (Cdc2) Metaphase/Anaphase (cohesin, APC) Cytokinesis (Ect2) G2 G1 M Plk1 CREST DAPI Merge Centrosomes Kinetochores Midzone Clone No.485 and No.509 produced positve results. Both Plk proteins binded with Polo-Box Domain. Below is a graphic of a Western Blot and the positve binding results. Kinetochores Centrosomes Isolation of Centrosome-Associated Polo-Box Domain-Binding Proteins Crystal Asamoah1,2,Anam Salman2, Nak-Kyun Soung2,Jung-Eun Park2, Kyung S. Lee2 University of North Texas1,National Cancer Institute/National Institutes of Health2 INTRODUCTION Objectives Scheme of experiment • Find out what proteins at the centrosome bind to Plk1. • What are the regulating effects of binding proteins? Protein Kinases belong to a vast family of enzymes, many of which play crucial roles in a wide range of cellular processes such as: cell division, proliferation, and apoptosis. Polo-like kinase 1 (Plk1) has been the focus of extensive studies because of its strong association with neoplastic transformation of human cells. Plk1 mRNA is overexpressed in a broad spectrum of human cancers such as breast, ovarian, colon, endometrial and esophageal carcinomas and leukemias. Plk1’s overexpression appears to be sufficient to override cellular checkpoints and induce genetic stability. The use of PLK1 inhibitors has increased our knowledge of mitotic regulation and has allowed us to asses their ability to suppress tumor growth in vivo. Materials and Methods Cloning A. PCR : cDNAtemplate, and phusion (NEB) DNA polymerase were used inorder to obtain a PCR product B. Digestion : Eco RV and Not I (NEB enzymes) C. Isolation of vector DNA and PCR product : A Qiagen gel extraction kit was used to perform isolation of digested vector and PCR product D. Ligation : Ligase (Takara) E. Transformation : Following ligation, the ligation products (plasmids) are transformed into bacteria for propagation, in our experiment we used XL10-Gold as competent cells. We then plated the competent cells onto LB+amp selective media to grow bacteria that included ligated plasmid. F. Select positive clones by digestion : Individual colonies are then afterward picked and tested for the wanted insert. Transfection : The desired orientation is transfected into 293T cells. The 293T cell was cell cultured using DMEM supplied with 10% FBS and 1% antibiotics, we subjected transfection into 293T cell using Lipofectamine 2000(invitrogen). GST Polo-Box Domain Pull down assay: We lysed the transfected 293T cells and made S15. Next, we mixed the S15 with GST -PBD WT or AM and incubated for 2 hours at 4˚C. Afterwards the beads were washed 5 times and then boiled after mixed with sample buffers. Immunoblotting We used HA antibody as a primary antibody and anti-mouse IgG, HRP-linked secondary antibody to detect the protein Polo-box domain Kinase Domain Plk1 PB1 PB2 Results Obje Conclusions • We have six positve clones, they are centrosomal proteins. • Among them, we found that Clone No.485 and No.509 binded with Polo-Box Domain and phosphorylation dependent manner. • From our research, These are novel Plk1 binding proteins. Elia, et al., 2003 Cell Cheng, et al., 2003 EMBO J. Garcia-Alvarez, et al., 2007 PNAS Consensus: [P/F]-[F/P]-[F/M]-[T/Q/H/M]-S-[pS/pT]-[P/X]-[F] S; invariable residue Stucture of Plk1 PBD and phospho-peptide complex Diagram of pCI-neo HA vector(new version) : we altered the MCS so that the new version includes HA tag and enzyme sites that are listed above. Refrences Yun SM et al., Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1 Nat Struct Mol Biol. 2009 Jul 13 Park JE et al., Direct quantification of polo-like kinase 1 activity in cells and tissues using a highly sensitive and specific ELISA assay. Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1725-30. Soung NK, et al., Plk1-dependent and -independent roles of an ODF2 splice variant, hCenexin1, at the centrosome of somatic cells. Dev Cell. 2009 Apr;16(4):539-50. Hanisch A, Wehner A, Nigg EA, Silljé HH. Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting. Mol Biol Cell. 2006 Jan;17(1):448-59. Strebhardt K, Ullrich A. Targeting polo-like kinase 1 for cancer therapy. Nat Rev Cancer. 2006 Apr;6(4):321-30 Above is a graphic of positive DNA cloning on electrophoresis gel. Multiple roles of Plk1 during M-phase progression