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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:

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

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  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

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