protein kinase analysis kinome. g protein-coupled receptor tyrosine kinase receptor ser/thr kinase...

Download Protein kinase analysis Kinome. G protein-coupled receptor Tyrosine kinase receptor Ser/Thr kinase receptor Cytokine receptor Intracellular Signal

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  • Protein kinase analysis Kinome
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  • G protein-coupled receptor Tyrosine kinase receptor Ser/Thr kinase receptor Cytokine receptor Intracellular Signal Transduction G AC cAMP PKA CREB PLC G Ca 2+ CaM CaMK DAG Sos Grb2 Ras Raf MEK ERK PKC Pi C-Myc Pi ELK Pi PI3K PKB FAK Pi Smad Pi JAK Pi STAT Pi Cyt-c Apaf-1 Casp-9 Casp-3 Bcl-2 Bad Pi GSK3 Pi Apoptosis Glycogen synthesis Adhesion Extension Transcription
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  • Protein kinases 1)ca. 500 kinds of putative kinases are coded in human genome 2) 1/3 of whole proteins are phosphorylated. 3)Certain protein kinase is hyper-activated in particular diseases, such as cancers, cardiovascular diseases Protein kinase signaling will be crucial target in diagnostics and drug discovery P + ATP+ ADP Protein kinases
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  • Disease cells and hyper-activated protein kinases Cancer Protein kinase C, Src, Akt, ALC, c-Met Inflammation I--kinase Cardiovascular diseases Rho kinase, Src Diabetes Protein kinase C
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  • Protein kinase Aqnti-phospho antibody Increase of the fluorescence polarization Protein kinase + Fluorescence-labeled peptide Protein substrate Increase of the fluorescence polarization a) b) Assay of protein kinase activity by using fluorescence polarization
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  • GFP Anti-phospho antibody GFP Time-resolved fluorescence assay using GFP fluorescence Protein kinase FRET a) fluoreophore quencherFRET Quenching of the fluorescence Protein kinase elastase Non-fluorescent elastase Recovery of the fluorescence b) Assay of protein kinase activity by using FRET
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  • GST Moesine LRRK2 GST Moesine Donor bead Acceptor bead GST Moesine h 1O21O2 Protein kinase Ga Quencher Quenching of the bead due to FRET with the quencher Other protein kinase assay -Screen Assay QTL assay
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  • Fluorescent probe for protein kinase using D-RECS strategy Polycation with substrate Polyanion labeled with fluoresceine Polyion complex (quenching) Recovery of fluorescence
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  • Protein kinase assay using Au nano-particle Protein kinase assay using Au nano-particle Phosphorylated substrate Substrate peptide
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  • B OD Inhibitor concentration (M) Phosphorylation ratio (%) OD Phosphorylation ratio OD 0 20 40 60 80 100 0 0.05 0.1 0.15 0.2 0.25 0.3 T umorNormal p- PKC actin A Normal (skin) Tumor B16 4 % 70 % 33 % 0 % p-PKC actin tumor normal inhibitor M - - 0.5 5.0 Inhibitor (Ro-31-7549) Detection of PKC activity in tumor tissue Normal tissue tumor tissue Tissue lysate Detected with Au-particle system
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  • Application to breast cancer prognosis Breast cancer (18 samples) Lysis of tissues PKC was detected with the assay P < 0.05 OD @ 670nm tumor normal 0 0.1 0.2 0.3 Biosensors Bioelectronics, 2010, 25, 1869. Normal tissue (12 samples) Low (6) Middle (6) High (6) Low Middle High
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  • Evaluation of kinase inhibitors J. Oishi et al, Anal. Biochem. 373, 161-163 (2008)
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  • Screening of PKA inhibitors 1234 5678 9101112 13141516
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  • Candidates found by the assay IC 50 8 11 H89 Staurosporine IC 50 (nM) calculated reported 95 4.8 144 7.0 Each compound was added in 0, 10, 30, 100, 300, 1000, 3000, 10000 nM to MCF-7 lysate, respectively and after the phosphorylation reaction, Au-particle dispersion was added to the reaction mixture. No. Name
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  • Cancer chemotherapy and molecular target drug 2/10 . problem with acquisition of resistance Some tumors acquire a resistances against molecular targeted drug with repeated administration. However, its mechanism has not been clarified. . survival benefit of molecular target drug Two drugs combination Avastin + Erbitax for colon cancer brought about 1.3 months shorter life expectancy comparing with the conventional anticancer drug 5- FU+OxPt) administration. Is molecular target drug also effective? (New England Journal of Medicine, 360, 563-572(2009) ) . survival benefit of cancer chemotherapy (Journal of the National Cancer Institute, 98, 1108-17(2006) ) When chemotherapy is used with operation, the survival benefit is just 5% for 5 years and only 1% for 15 years comparing with operation only Is cancer chemotherapy really effective Molecular target drug Drug which is effective only to target cell
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  • 19 Pathways in Human Cancer From R.A. Weinberg Appendix: The Biology of Cancer (Garland Science 2007, Taylor & Francis)
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  • 22 Human Kinome TK tyrosine kinase TKL tyrosine kinase like STE homologs of yeast sterile 7,11,20 CK1 casein kinase-1 AGC members of protein kinase A, G, C families CAMK Ca+ calmodulin-dependent protein kinases CMGC containing CDK, MAPK, GSK-3 and CLK families From R.A. Weinberg: The Biology of Cancer, Fig.16-12 (Garland Science 2007, Taylor & Francis)
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  • Peptide array for kinome 23 Peptide array: 30L / assay 1/2000 384 plate: 80mL / assay
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  • A B Cell lysate Phosphorylation pattern Profile A Profile B Normal cell Disease or drug administered cell Diagnostics based on the profile Drug discovery using the profile Concept of kinome profiling
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  • PepChip Array Each peptide is immobilized in micro-well 50 L 1152 kinds of peptides can be immobilized Detect with 33 P-ATP PamChip Array Substrates are immobilized on a porous support Repeat the putting in and out repeatedly to promote the reaction Detect with 33 P-ATP 144 kinds of substrate can be immobilized. Barretts esophagus green acive in BE red inactive in BE MAPK signaling was suppressed and glycolytic metabolism was activated in BE Commercial available kinome arrays
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  • CelluSpot Array Each substrate is synthesized on a cellulose membrane (SPOT synthesis) Radius of each spot: 1.2 mm Detect with HRP-labeled anti-phosphotyrosine antibody (CL) 384 kinds of peptides can be immobilized.
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  • Our peptide array Phos-tag Cy3- streptavidin Phos-tag PPPP PPPP PPPP Detect with Microarray scanner
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  • Surface chmistry cover Phosphorylation P Y Y Y
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  • Application to Iressa sensitivity EGFR AktERK iressa HCC827 EGFR AktERK A549 Highly sensitive EGFR kinase with mutation Resisitant Other pathway is activated. Other receptors iressa Cell deathCell survive
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  • Protocol Iressa 2 hour EGF 15min phosphorylation 3 hour phostag-SA 1000 peptides lysis HCC827 IC 50 =16 nM A549 IC 50 =30 M [Iressa] 3 nM300 nM
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  • Comparison between A549 and HCC827 Iressa inhibited the phosphorylation of many substrate in HCC827. Iressa(-) iressa 300 nM A549 HCC827 Tyr peptide Ser/Thr peptide Tyr peptide Iressa(-) iressa 300 nM
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  • Alternative pathway is Src-pathway? A549HCC827 iressa(+)/iressa(-) EGFR Src EGFR Src
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  • peptide No. activation suppression Kinome Profile modification with metformin (Dr. Osamu Okitsu) ID activation suppression Drug A


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