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. Nahshan St.Bernard Yun Yong Park Hueng-Sik Choi Barry University Hormone Research Center Chonam National university. Nuclear Receptors (NRs). largest family transcription factors

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cross talk between orphan nuclear receptor shp and jak stat pathway mediated transcription

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

nuclear receptors nrs
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
short heterodyning partner shp
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
jak stat pathway
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)
jak stat pathway8
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
slide10

Transcription

Target gene

aim of study
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
slide12

- Cloning of STAT gene

Cloned using PCR, and then transferred into a E.coli

STAT

STAT

gst pull down
GST Pull Down
  • Produced SHP protein via in vitro

transcription

  • Transfected cells
gst pull down assay
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.
slide15

- GST pull down to confirm the interaction SHP and STAT

SHP

STAT

Protein

Induction

In vitro

translation

slide16

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

does shp have an inhibitory effect on stat expression in hela cell line
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

slide19

STAT and SHP(Hela cell)

M67

+ + + + + + + + + + + + + + + +

- + + + - + + + - + + + - + + +

- - + + - - - - - - - - - - - -

- - - - - - + + - - - - - - - -

- - - - - - - - - - + + - - - -

- - - - - - - - - - - - - - + +

- - - + - - - + - - - + - - - +

Tel.Jak2

STAT 1

STAT` 3

STAT 5a

STAT 5b

mSHP

(200ng)

the effects of various concentrations of shp on stat 1 and 3
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

slide21

(HepG2 cell)

M67

  • + + + + + + + + + + + +
  • - + + + + + - + + + + +
  • - - + + + + - - - - - -
  • - - - - + - - + + + +
  • - - 100 300 600 - - - 100 300 600

Tel.Jak2

STAT 1

STAT 3

SHP

discussion
Discussion
  • The levels of STAT were not

repressed significantly by SHP

What does mean?