A Novel Mechanism for JAK2 Activation by a G Protein-Coupled Receptor, the CCK2R. “Implication of this Signaling Pathway in Pancreatic

Tumor Models”.  Ferrand et al. (2005). Journal of Biological Chemistry

Presented by:

Salina Gairhe

Bio 4751

Background

G Protein-coupled Receptors:

7 transmembrane domain ligand binds to the extracellur portion of receptor activate an adjacent G protein stimulates second messenger mediated cascades second messengers can activate broad and diverse target

proteins in the cell

Diversity of G Protein-Coupled Receptor Signal transduction Pathways

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Cholecystokinin receptors (CCKR)

Types: CCK1R and CCK2R CCK1R : associated with regulation of food

intake/endocrine regulation CCK2R: initially thought to be associated with secretory

effect of gastrin activates various mitogenic signaling pathways when

stimulated by gastrin on gastrointestinal and pancretic acinar cells

increases proliferation of normal and neoplastic gastrointestinal cells

Janus Activating Kinase

JAK family of intracellular tyrosine kinases consists of Jak1, Jak2, Jak3, and Tyk2, which range in size from 130 to

135 kDa.Janus Activating Kinase 2 (JAK2) ---is a 130 kDa tyrosine kinase ---involved in cytoplasmic signal transduction. ---mainly in cancerMecahnism: Ligand binds to a variety of cell surface receptors (e.g., cytokine, growth

factor, GPCRs) leads to an association of those receptors with JAK proteins which are then activated via phosphorylation on tyrosines 1007 and 1008

in the kinase activation loop.

STAT:

• Signal Transducer and Activators of Transcription• proteins and are inactive as a monomer• activation involves phosphorylation and dimerization• previously, they were found to be activated by Cytokine receptors • Now, a wide variety of ligands are found to activate STAT family

members. • Among the STAT family members activated by JAKs

kinases,STAT-3 is recognized as oncogenes• involved in various biological functions like cell transformation,

development, differentiation, immunity, and apoptosis.

Structure:

STATs possess a single highly conserved tyrosine phosphorylation site,

1. SH2 domain: receive signal from tyrosine kinase such as Jak 2. DNA-binding domain: activate transcription

DNA binding SH3 SH2

JAK-STAT PathwayReceptors single-pass transmembrane proteins embedded in the plasma

membrane. Ligands inteferon interleukinsMechanisms Binding of the ligand causes two receptors to form a dimer. The dimer activates a Janus kinase ("JAK") which phosphorylates certain tyrosine (Tyr) residues on one or another of

several STAT proteins These, in turn, form dimers which enter the nucleus and bind to

specific DNA sequences in the promoters of genes that begin transcription.

The JAK-STAT pathways are much shorter and simpler than the pathways triggered by RTKs and so the response of cells to these ligands tends to be much more rapid.

JAK/STAT pathway contd…..

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These pathways when deranged, lead to cancer and other harmful effect on cell

GOAL

To analyze the mechanism for Jak-2 activation by the CCK2R

To determine the putative role of this signaling pathway in the pathophysiological functions of this receptor in pancreatic tumor models

Epidemology

Pancreatic carcinoma is fourth leading cause of cancer

Each year 30,000 cases are diagnosed, 95% die within 5 year

(National Cancer Institute) no adequate therapy

Pancreatic cancer arises from oncogenic transformation of pancreatic ductal epithelial cells

Gastrin

acinar cells of pancreas secrets the GASTRIN regulator of gastric acid secretion. has an important growth-promoting influence on the

gastric mucosa gastrin receptor binds cholecystokinin, and is known as

the CCK-B receptor. member of the G q protein-coupled receptor family.

binding of gastrin stimulates an increase in intracellular Ca++, activation of protein kinase C, and production of inositol phosphate

Experimental Procedures

Animal: Elas-CCK2 mice Immunohistochemistry Western Blot Analysis on Isolated

Acinar Cells or AR4–2J Cells Cell Culture and Proliferation Assay JAK2 Kinase Assay Construction of Mutant Receptor

cDNAs and Transient Transfection Measurement of Inositol Trisphosphate

(IP3 ) Accumulation Immunofluorescence Staining

Animal

Transgenic Elas-CCK2 mice

targeted for expression of human CCK2 receptor in pancreatic acinar cells

by using transcriptional element of the elastase-1 gene mice exhibit an increased pancreatic growth ,an acinar to

ductal trans-differentation postulated to be preneoplastic step in pancratic

carcinogenesis and tumor development

Immunohistochemistry Pancreatic tissues were collected and analyzed for the activation of

JAK2 and STAT3 by CCK2R receptors in acini of ELAS-CCK2 mice following steps:

primary antibody binds to specific antigen

antibody-antigen complex is bound by a

secondary, enzyme-conjugated, antibody;

in the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen

binding.

10 Ab Ag

10 AbAg

20 AbHRP

Ab Ag

Western Blot Analysis acinar cell lysates antibodies specific for

total JAK2 (JAK2) and STAT3(STAT3)

To detecting the active form of the protein, anti-PY1007–1008 JAK2 antibodies (PY-JAK2) and anti-PY705 antibodies (PY- STAT3)

blots were also probed with an antibody against

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to ensure equal

loading of proteins

Cell Culture and Proliferation Assay

ARJ4-2J cells only pancreatic tumor cell line exhibiting an acinar

phenotype, established after a chemo-induced tumorigenesis by azaserine

previously shown to express an endogenous CCK2R

Procedure: cells were plated for culture and proliferation serum starved for 24hrs treated with gastrin for 48hrsTo monitor the CCK2 activated cell proliferation by JAK-2,

cells were incubated with AG490, a JAK2 specific inhibitor

JAK2 Kinase Assay

1. cells were stimulated with gastrin cells and lysed

2. cell lysate was immunoprecipitated with anti-PY1007–1008 JAK2 antibody (UBI) for JAK2

3. pellet were suspended in 1X kinase buffer supplemented with ATP and substrate.

4. incubate 30 minutes at 30°C.

5. reaction was terminated with SDS sample buffer

6. vortex, then was centrifuged for 30 seconds

7. sample were heated at 95–100°C for 2–5 minutes

8. sample was loaded on SDS-PAGE gel

9. sample was analyzed by Western blotting

Construction of Mutant Receptor cDNAs and Transient Transfection

Construction of Mutant Receptor cDNAs

Mutant receptor cDNAs were constructed by oligonucleotide-directed mutagenesis using the rat CCK2R

cDNAs were subcloned in prk5-JAK2 vector mutations were confirmed by DNA sequencing using an automated

sequencer  

 Transfection of Wild Type and Mutant Receptor cDNAs into Mammalian Cells

COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. 2 µg of plasmids coding for wild type CCK2R (WT-CCK2R) or mutant

CCK2R for JAK2 or for HA-tagged q (Q209L) mutant were transiently transfected into COS-7 cells using the DEAE/dextran

Measurement of total IP accumulation

after 24hrs of COS-7 cell transfection, the transfected cells were transferred to 24-well culture plates

incubated overnight in DMEM with 3 µCi/well of myo-2-[3H]inositol

incubated 1 hr at 37° with IP buffer containing the indicated concentrations of CCK2R

reaction was stopped with 1 ml of methanol/HCl added to each well and content transferred to a Dowex AG 1-X8 (formate form) column

each column was washed with 5 ml of water followed by 2 ml of 5 mM sodium tetraborate/ 60 mM sodium formate.

total IP were eluted from the columns with 2 ml of 1 M ammonium formate/100 mM formic acid

total [3H]IP -radioactivity was detected in a liquid scintillation counter

Immunofluorescence Staining

COS-7 cells were grown for 24hrs on plates containing cover slides.

cells were fixed in 2% paraformaldehyde,

After 24 h of transfection:

cells were permeabilized with methanol blocked in 1% fetal calf serum-phosphate-buffered saline, incubated with primary antibodies (anti-HA antibody

(Berkeley Antibody Co., Covance), anti-PY1007–1008 JAK2 antibody (UBI)

according to standard immunofluorescence methods. secondary antibodies coupled to CY-3 or fluorescein

Main questions to be addressed

1. Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2?

2. Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT-3?

3. Dose CCR2R activates JAK2 in AR4-2J cells?4. Dose CCR2R activates STAT-3 in AR4-2J cells?5. Is there any role of JAK2 in Acinar Tumor Cell

Proliferation Induced by CCK2R Activation?6. Does CCK2R in COS-7 cells activates JAK2?7. Is there involvement of NPXXY motif in

activation of JAK2/STAT3 pathway by the CCK2R?

8. Is there any role of G protein in JAK2 activation?

Q.1 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2?

Control Elas-CCK2

ControlElas-CCK2

PY-JAK2 PY-JAK2

Ans: The expression of the CCK2R in mouse pancreatic acini induces JAK2 activation

Immunohistochemical methods

Western blotting

PY-JAK2: protein phosphorylated on tyrosines 1007 and 1008

Q.1 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2?

Control Elas-CCK2

JAK-2 JAK-2

Immunohistochemical methods:

In contrast, total JAK2 protein expression was unchanged in the two mice models.

Q.2 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT3?

Control Elas-CCK2

PY-STAT-3 PY-STAT-3

Control Elas-CCK2

Ans: The expression of the CCK2R in mouse pancreatic acini induces STAT-3 activation

Immunohistochemical methods

Western blotting

P Y-STAT-3: protein phosphorylated on tyrosine 705

Q.2 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT3?

Control Elas-CCK2

In contrast, total STAT-3 protein expression was unchanged in the two mice models.

STAT-3 STAT-3

Q.3 Dose CCR2R activates JAK2 in AR4-2J cells?

Gastrin significantly activated JAK2 in AR4–2J cells after serum starvation

A rapid and significant increase in JAK2 phosphorylation at 1 min that was maximal after 1 h of stimulation by the CCK2R agonist(AG490)

Ans: CCR2R activated JAK2 in AR4-2J cells

Jak-2 Kinase Assay autophosphorylation of JAK-2 by P-JAK-2

IB=Western blotting

Q.4 Dose CCR2R activates STAT3 in AR4-2J cells?

•CCK2R induced significant activation of STAT3 from 15 to 120 min after gastrin treatment• with a maximal activation after 60 min

•amount of total STAT3 protein remains unchanged

To determine the involvement of JAK2 in CCK2R-induced STAT3 activation,• the effect of a JAK2 inhibitor AG490, on STAT3 activation was tested in response to gastrin

•phosphorylation of STAT3 after gastrin stimulation was completely blocked by the JAK2 inhibitor

•Thus, CCK2R-induced STAT3 activation is totally JAK2-dependent in this cellular model.

IB=Western blotting

Ans: CCR2R activated STAT-3 in AR4-2J cells

Q.5 Is there any role of JAK2 in Acinar Tumor Cell Proliferation Induced by CCK2R activation?

To study the role of JAK-2 in the proliferation of acinar tumor cells induced by the CCK2R

• AR4–2J proliferation were measured in the presence or absence of the Jajk-2 specific inhibitor after 48 h of gastrin stimulation

• CCK2R activation by gastrin induces a significant increase of cell proliferation

• Treatment of the cells with AG490 totally inhibits CCK2R-inducedR4–2J proliferation. This result confirms that JAK2 mediates CCK2R proliferative effects on AR4–2J cells.

Ans: JAK2 mediates CCK2R proliferative effects on AR4–2J cells.

Q.6 Does CCK2R in COS-7 cells activates JAK2?

To study the molecular mechanism involved in JAK2 activation by the CCK2R, COS-7 cells were used

• Using JAK2 autophosphorylzation ability, in vitro Tyrosine Kinase Assays were performed in anti-JAK2 immunoprecipitates from cell lysates

• Gastrin significantly activated JAK2 in this transiently transfected cell model

• rapid and significant activation of JAK2 (15 s),

• Still detectable at 3 min, was found in response to gastrin

• Western blot analysis for JAK2 protein expression revealed an equal amount of the protein in transfected cells.

Ans: CCK2R in COS-7 cells activates JAK2

IB=Western blotting

Q.7 Is NPXXY motif involved in activation of JAK2/STAT3 pathway by the CCK2R?

Fig. 7, A and B

N386A-CCK2R mutant cannot mediate JAK2 activation, in contrast to the WT-CCK2R.

Fig. 7C

• the activation of the downstream effector of JAK2, STAT3, is also blocked when the CCK2R is mutated on the NPXXY motif

Ans: NPXXY motif within the receptor sequence is required for the activation of the JAK2/STAT3 pathway by the CCK2R

IB=Western blotting

Jak-2 Kinase Assay

Q.8 Are G q proteins involved in JAK2 activation?

Immunofluorescence study

• solid arrow shows cells strongly expressing the q constitutive mutant display a high level of staining for the activated JAK2 protein, very dotted arrow shows weak staining appears in COS-7 cells showing a low HA-tagged protein expression

• JAK2 activation by using the anti-PY1007–1008 JAK2 antibody

Q8. Are G proteins involved in JAK2activation?

Ans:G proteins are involved in JAK2activation

• to show association between G q and JAK2 in COS-7 cells transfected (T) or not (NT) constitutively activated q mutant.

•Cell lysates were immunoprecipitated (IP) with an anti-HA antibody and Western-blotted with the anti-JAK2 antibody.

NT: COS7 cell not transfected with the c DNA CDNA coding for the HA-tagged(Q209L)

T: COS7 cell transfected with the c DNA CDNA coding for the HA-tagged(Q209L)

IB=Western blotting

Conclusion

a new mechanism was discovered in activation of JAK2 involving G q protein

this mechanism is used by the CCK2R to activate tyrosine kinase and involved the NPXXY motif within the receptor sequence.

Both in vitro and in vivo pancreatic models, the CCK2R activated the JAK2/STAT3 signaling pathway,a transduction cascade up-regulated during the tumor process in human

Reference Ferrand et al (2005). A Novel Mechanism for JAK2 Activation by

a G Protein-Coupled Receptor, the CCK2R. Implication of this Signaling Pathway in Pancreatic Tumor Models. Journal of Biological Chemistry. Vol. 280, No. 11, Issue of March 18, pp. 10710–10715, 2005

Marchal et al (2002). Genetic, pharmacological and functional analysis of cholecystokinin-1 and cholecystokinin-2 receptor polymorphism in type 2 diabetes and obese patients. Pharmacogenetics. 12(1):23-30, January

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