CROSS TALK BETWEEN ORPHAN NUCLEAR RECEPTOR SHP AND JAK/STAT PATHWAY MEDIATED TRANSCRIPTION - PowerPoint PPT Presentation

cross talk between orphan nuclear receptor shp and jak stat pathway mediated transcription l.
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CROSS TALK BETWEEN ORPHAN NUCLEAR RECEPTOR SHP AND JAK/STAT PATHWAY MEDIATED TRANSCRIPTION PowerPoint Presentation
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CROSS TALK BETWEEN ORPHAN NUCLEAR RECEPTOR SHP AND JAK/STAT PATHWAY MEDIATED TRANSCRIPTION

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  1. CROSS TALK BETWEEN ORPHAN NUCLEAR RECEPTOR SHP AND JAK/STAT PATHWAY MEDIATED TRANSCRIPTION Nahshan St.Bernard Yun Yong Park Hueng-Sik Choi Barry University Hormone Research Center Chonam National university

  2. Nuclear Receptors (NRs) • largest family transcription factors • approximately 50 individual family members in mammals. • hormonal and metabolic control of transcription • involved in physiology, disease, cancer regulation, cell proliferation, differentiation, and development • ligand-activated • regulates gene expression • includes a large number of orphan receptors for which have no ligand

  3. Short Heterodyning Partner (SHP) • Not a typical NR; does not contain DNA Binding Domain (DBD) • Expressed in Liver, Spleen, Small Intestines, and Pancreas as wells as other tissue types and organs • Known repressor of transcription of a number of NRs

  4. JAK/ STAT PATHWAY • Janus Active Kinase • Cytoplasmic protein kinase • Kinase: catalyzes the transfer of phosphate groups from a high-energy phosphate-containing molecule to a substrate • Activated by cell surface receptors • Catalyzes activation of Signal Transducer and Activator of Transcription (STAT)

  5. JAK/ STAT PATHWAY • Signal Transducer and Activator of transcription • Activated by interferons, a part of the Cytokines family of molecules • Cytokines are a large family of extracellular ligands • Stimulatebiological responses in virtually all cell types • There are seven stat genes, STAT; 1,2,3,4,5a,5b,6 • Play a role in cancer regulation

  6. Transcription Target gene

  7. Aim of Study - Identification of cross talk between ONR(SHP) and transcription factor(STAT) Experimental Approach • Confirm the interaction between SHP and STAT family using a Yeast two hybrid and GST pull down • Functional assay that SHP affects trans-activation of STAT

  8. - Cloning of STAT gene Cloned using PCR, and then transferred into a E.coli STAT STAT

  9. GST Pull Down • Produced SHP protein via in vitro transcription • Transfected cells

  10. GST Pull Down Assay • In vitro technique that consists of a fusion tagged bait protein for which a binding partner is being sought • The gst-bait is bound to an immobilized gluthanione support or a his-tagged is bound to a metal chelate support • the immomiblized protein is introduced to a protein pool derived from a cell lysate. After the washing steps the interacting proteins are eluted selectively and are then detected in a gel.

  11. - GST pull down to confirm the interaction SHP and STAT SHP STAT Protein Induction In vitro translation

  12. Transfection to confirm trans-activation between SHP and STAT Grow the cell line (Hela, HepG2)and seed co- transfect with STAT,SHP and Tel. Jak2 Lyses the cell and measure the activity Incubate for 2 days

  13. Results

  14. Does SHP Have an inhibitory effecton STAT expression in Hela cell line? Compared the expression of an internal control M67 with Tel. JAK and the various STAT molecules

  15. STAT and SHP(Hela cell) M67 + + + + + + + + + + + + + + + + - + + + - + + + - + + + - + + + - - + + - - - - - - - - - - - - - - - - - - + + - - - - - - - - - - - - - - - - - - + + - - - - - - - - - - - - - - - - - - + + - - - + - - - + - - - + - - - + Tel.Jak2 STAT 1 STAT` 3 STAT 5a STAT 5b mSHP (200ng)

  16. The Effects of Various Concentrations of SHP on STAT 1 and 3 Stats 1 and 3 were used because of their high expression levels in various cancers and tumors

  17. (HepG2 cell) M67 • + + + + + + + + + + + + • - + + + + + - + + + + + • - - + + + + - - - - - - • - - - - + - - + + + + • - - 100 300 600 - - - 100 300 600 Tel.Jak2 STAT 1 STAT 3 SHP

  18. Discussion • The levels of STAT were not repressed significantly by SHP What does mean?