1 / 37

Ferrand et al. (2005). Journal of Biological Chemistry

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:

linus-vasquez
Télécharger la présentation

Ferrand et al. (2005). Journal of Biological Chemistry

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. 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

  3. Diversity of G Protein-Coupled Receptor Signal transduction Pathways Source:http://www.sigmaaldrich.com

  4. 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

  5. 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 cancer Mecahnism: • 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.

  6. 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, • SH2 domain: receive signal from tyrosine kinase such as Jak • DNA-binding domain: activate transcription DNA binding SH3 SH2

  7. JAK-STAT Pathway Receptors • single-pass transmembrane proteins embedded in the plasma membrane. Ligands • inteferon • interleukins Mechanisms • 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.

  8. JAK/STAT pathway contd….. Source:http://www.sigmaaldrich.com

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

  10. 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

  11. 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

  12. Pancreatic cancer arises from oncogenic transformation of pancreatic ductal epithelial cells

  13. 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

  14. 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

  15. 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

  16. 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 Ab Ag 20 Ab HRP Ab Ag

  17. 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

  18. 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 48hrs To monitor the CCK2 activated cell proliferation by JAK-2, cells were incubated with AG490, a JAK2 specific inhibitor

  19. JAK2 Kinase Assay • cells were stimulated with gastrin cells and lysed • cell lysate was immunoprecipitated with anti-PY1007–1008 JAK2 antibody (UBI) for JAK2 • pellet were suspended in 1X kinase buffer supplemented with ATP and substrate. • incubate 30 minutes at 30°C. • reaction was terminated with SDS sample buffer • vortex, then was centrifuged for 30 seconds • sample were heated at 95–100°C for 2–5 minutes • sample was loaded on SDS-PAGE gel • sample was analyzed by Western blotting

  20. 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

  21. 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

  22. 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 antibodiescoupled to CY-3 or fluorescein

  23. Main questions to be addressed • Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2? • Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT-3? • Dose CCR2R activates JAK2 in AR4-2J cells? • Dose CCR2R activates STAT-3 in AR4-2J cells? • Is there any role of JAK2 in Acinar Tumor Cell Proliferation Induced by CCK2R Activation? • Does CCK2R in COS-7 cells activates JAK2? • Is there involvement of NPXXY motif in activation of JAK2/STAT3 pathway by the CCK2R? • Is there any role of G protein in JAK2 activation?

  24. Q.1 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2? PY-JAK2: protein phosphorylated on tyrosines 1007 and 1008 Elas-CCK2 Control Immunohistochemical methods PY-JAK2 PY-JAK2 Western blotting Control Elas-CCK2 Ans: The expression of the CCK2R in mouse pancreatic acini induces JAK2 activation

  25. Q.1 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of JAK2? Immunohistochemical methods: Elas-CCK2 Control JAK-2 JAK-2 In contrast, total JAK2 protein expression was unchanged in the two mice models.

  26. Q.2 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT3? P Y-STAT-3: protein phosphorylated on tyrosine 705 Elas-CCK2 Control Immunohistochemical methods PY-STAT-3 PY-STAT-3 Western blotting Control Elas-CCK2 Ans: The expression of the CCK2R in mouse pancreatic acini induces STAT-3 activation

  27. Q.2 Dose CCR2R expression in acini of ELAS-CCK2 mice induces the activation of STAT3? Control Elas-CCK2 STAT-3 STAT-3 In contrast, total STAT-3 protein expression was unchanged in the two mice models.

  28. Q.3 Dose CCR2R activates JAK2 in AR4-2J cells? Jak-2 Kinase Assay autophosphorylation of JAK-2 by P-JAK-2 IB=Western blotting 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

  29. 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

  30. 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.

  31. Q.6 Does CCK2R in COS-7 cells activates JAK2? IB=Western blotting 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

  32. Q.7 Is NPXXY motif involved in activation of JAK2/STAT3 pathway by the CCK2R? Jak-2 Kinase Assay IB=Western blotting 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

  33. Q.8 Are G q proteins involved in JAK2 activation? Immunofluorescence study • JAK2 activation by using the anti-PY1007–1008 JAK2 antibody • 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

  34. Q8. Are G proteins involved in JAK2activation? IB=Western blotting • 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) Ans:G proteins are involved in JAK2activation

  35. 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

  36. 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

  37. Thank You

More Related