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Protein Interaction Analysis Applications Presented Fall 2009 Click the icon in the upper left hand corner to view speaker notes for slides. Have a question? Ask a Scientist DLR and CheckMater are trademarks and Dual-Luciferase and Flexi are registered trademarks of Promega Corporation. HighWire Press is a registered trademark of the Board of Trustees of the Leland Stanford Junior University ©2009, Promega Corporation. All rights reserved. Application Overview POST-TRANSCRIPTION MIRNA CONTROL PROTEIN In vivo applications INTERACTIONS NUCLEAR RECEPTORS SIGNALING PATHWAY PROMOTER DISSECTION 2 expression level varies with treatment Experimental firefly luciferase construct expression level varies little with treatment Control Renilla luciferase construct Do these proteins interact in mammalian cells? A yeast two-hybrid study says these two proteins interact. Do they still interact in a mammalian background? Reporter assays can provide a rapid method to confirm interactions and even a way to investigate the interactions in more detail. 3 Exploiting Transcription Factor domains for protein interaction studies BD RNA Pol II AD TF Binding Domain Exon TATA Box Exon Exon • Transcription Factors have a DNA-binding domain (BD) that brings it next to the gene and a transcription activation domain (AD) that complexes with RNA Polymerase II to begin transcription Prey Bait GAL4 BD BD TF GAL4-GAL4-GAL4 Binding Domain AD VP16 RNA Pol II RNA Pol II TATA Box Exon Exon Reporter Gene Exon Exon 1st used for Yeast Two-Hybrid Assays to identify protein:protein interactions Exon • Removal/mutagenesis of either the binding domain or the activation domain yields a non-functional transcription factor • You can bridge a binding domain and an activation domain with two proteins that interact and activate transcription. 4 CheckMate uses Yeast Two-Hybrid principles in Mammalian cells Prey pBIND Vector GAL4 UAS 1 TRANSFECT Bait VP16 GAL4 BD GAL4 UAS GAL4 UAS GAL4 UAS TATA Box luc2 2 CULTURE 2-3 DAYS 3 pGL4.31 DUALLUCIFERASE® ASSAY pGL4.31 100% pBIND/pACT-Raf Relative Expression Ratio GAL4 UAS pACT Vector 80% pBIND-Ras/pACT pBIND-Ras/pACT-Raf 60% 40% 20% pACT pBIND 0% 24 hours 48 hours 72 hours Post Transfection 5 Case Study: cdk3 interaction with ATF1 • cdk3 highly expressed in glioblastoma tissues and cell lines • cdk3 is a ser/thr kinase • Could the cdk3 be targeting transcription factors in the glioblastomas? • Utilized the CheckMate System to find the answers. Zheng, D., et al. (2008) Cancer Res. 68, 7650-60. 6 Case Study: Zheng, D., et al. ATF1:cdk3 found with CheckMate System CheckMate System used to explore whether or not cdk3 interacted with selected transcription factors pG5-luc pACT TF’s pBIND cdk3 Transcription Factor Result E2F1 (– control) basal activity E2F2 (+ control) >3-fold increase ATF1 >15-fold increase ATF2 basal activity ATF3 basal activity STAT2 basal activity Interaction confirmed by immunoprecipitation & cdk3 phosphorylates ATF1 Ser63 7 More Information Protocols & Applications Guide: Protein:Protein Interaction Analysis: In Vivo and In Vitro Methods Go to CheckMate™ Technical Manual Schenborn, E., et al. (2006) The nextgeneration assay for mammalian protein interactions: The CheckMate™/Flexi® Vector Mammalian Two-Hybrid System. Promega Notes 94, 12-16. CheckMate™ System citations from HighWire Press® CheckMate™ System citations from Nature.com 8 Please note, the CheckMate™/Flexi® Vector Mammalian Two-Hybrid System is an improved version of the original CheckMate™ Mammalian Two Hybrid System. The original pBIND and pACT vectors are in the CheckMate™ Negative Control Vector Set