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Protein kinase analysis Kinome

Protein kinase analysis Kinome. PKA. G protein-coupled receptor. STAT. Smad. ELK. CREB. C-Myc. CaM. Ras. Apaf-1. PLC. Casp-3. Bcl-2. Casp-9. Bad. Cyt-c. Grb2. G. G. Sos. Ca 2+. Tyrosine kinase receptor. cAMP. DAG. AC. JAK. GSK3. CaMK. Raf. MEK. FAK. ERK. PI3K.

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Protein kinase analysis Kinome

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  1. Protein kinase analysis Kinome

  2. PKA G protein-coupled receptor STAT Smad ELK CREB C-Myc CaM Ras Apaf-1 PLC Casp-3 Bcl-2 Casp-9 Bad Cyt-c Grb2 G G Sos Ca2+ Tyrosine kinase receptor cAMP DAG AC JAK GSK3 CaMK Raf MEK FAK ERK PI3K PKB PKC Apoptosis Transcription Adhesion Extension Glycogen synthesis Cytokine receptor Ser/Thr kinase receptor Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi Intracellular Signal Transduction

  3. P Protein kinases + ATP + ADP 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

  4. Disease cells and hyper-activated protein kinases Cancer：Protein kinase Cα, Src, Akt, ALC, c-Met Inflammation：I-κ-kinase Cardiovascular diseases：Rho kinase, Src Diabetes：Protein kinase Cβ

  5. Assay of protein kinase activity by using fluorescence polarization a) Aqnti-phospho antibody Protein kinase Fluorescence-labeled peptide Increase of the fluorescence polarization b) Protein kinase + Protein substrate Increase of the fluorescence polarization

  6. Assay of protein kinase activity by using FRET a) FRET Anti-phospho antibody Protein kinase GFP GFP GFP Time-resolved fluorescence assay using GFP fluorescence b) quencher FRET fluoreophore elastase Protein kinase Quenching of the fluorescence Non-fluorescent elastase Recovery of the fluorescence

  7. Other protein kinase assay a-Screen Assay 1O2 hn Moesine Moesine Donor bead LRRK2 Moesine GST GST Acceptor bead GST QTL assay Quencher Protein kinase Ga Ga Quenching of the bead due to FRET with the quencher

  8. Fluorescent probe for protein kinase using D-RECS strategy Polycation with substrate Polyanion labeled with fluoresceine Polyion complex (quenching) Recovery of fluorescence

  9. Protein kinase assay using Au nano-particle Phosphorylated substrate Substrate peptide

  10. A Detection of PKCαactivity in tumor tissue B Normal(skin)Tumor（B16） 100 Phosphorylation ratio inhibitor（mM） - - 0.5 5.0 80 OD 60 p-PKCα 40 actin 0.3 20 0 . 25 4 % 70 % 33 % 0 % 0 0 . 2 tumornormal OD 0 . 15 p-PKCa Phosphorylation ratio (%) 0 . 1 actin Normal tissue tumor tissue 0 . 05 0 Tumor Normal OD Tissue lysate Detected with Au-particle system Inhibitor (Ro-31-7549) Inhibitor concentration (μM)

  11. Application to breast cancer prognosis P < 0.05 0.3 Low (6) 0.2 Middle (6) OD @ 670nm 0.1 High (6) Breast cancer (18 samples) Normal tissue (12 samples) 0 tumor normal Lysis of tissues High PKCawas detected with the assay Middle Low Biosensors Bioelectronics, 2010, 25, 1869.

  12. Evaluation of kinase inhibitors J. Oishi et al, Anal. Biochem. 373, 161-163 (2008)

  13. Screening of PKA inhibitors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

  14. Candidates found by the assay －IC50－ Each compound was added in 0, 10, 30, 100, 300, 1000, 3000, 10000 nMto MCF-7 lysate, respectively and after the phosphorylation reaction, Au-particle dispersion was added to the reaction mixture. IC50 (nM) No. Name calculated reported 8 11 H89 Staurosporine 95 4.8 144 7.0

  15. Ｋｉｎｏｍｅ　＆　Ｋｉｎｏｍｉｃｓ

  16. Cancer chemotherapy and molecular target drug １. survival benefit of cancer chemotherapy Molecular target drug Drug which is effective only to target cell 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？ (Journal of the National Cancer Institute, 98, 1108-17(2006)より ) ２. 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)より) ３. problem with acquisition of resistance Some tumors acquire a resistances against molecular targeted drug with repeated administration. However, its mechanism has not been clarified. 2/10

  17. Pathways in Human Cancer From R.A. Weinberg Appendix: The Biology of Cancer (Garland Science 2007, Taylor & Francis)

  18. 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 Human Kinome From R.A. Weinberg: The Biology of Cancer, Fig.16-12 (Garland Science 2007, Taylor & Francis)

  19. Peptide array for kinome 384 plate: → 80mL/ assay Peptide array: 30µL / assay 1/2000分

  20. パターンA パターンB Concept of kinome profiling Phosphorylation pattern Cell lysate Profile A Normal cell Diagnostics based on the profile Drug discovery using the profile Cell lysate Disease or drug administered cell Profile B

  21. Commercial available kinome arrays PepChip Array Each peptide is immobilized in micro-well（50μL） 1152 kinds of peptides can be immobilized Detect with 33P-ATP PamChip Array Substrates are immobilized on a porous support Repeat the putting in and out repeatedly to promote the reaction Detect with 33P-ATP 144kinds of substrate can be immobilized. Barrett’s esophagus green：acive in BE red：inactive in BE ・MAPKsignaling was suppressed and glycolyticmetabolism was activated in BE

  22. 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.

  23. Our peptide array P P P P 細胞破砕液 Phos-tag Phos-tag P P P P P P P P Cy3-streptavidin Detect with Microarray scanner

  24. Surface chmistry Y P Y Y cover Phosphorylation

  25. Application to Iressa sensitivity A549 HCC827 ・Resisitant ・Other pathway is activated. ・Highly sensitive ・EGFR kinase with mutation EGFR Other receptors EGFR iressa iressa Akt ERK Akt ERK Cell death Cell survive

  26. Protocol 3 nM 300 nM [Iressa] HCC827 IC50=16 nM A549 IC50=30μM EGF 15min lysis Iressa 2 hour phosphorylation 3 hour phostag-SA 1000 peptides

  27. Comparison between A549and HCC827 HCC827 A549 Tyr peptide Ser/Thr peptide Tyr peptideのうち良く反応 iressa 300 nM iressa 300 nM Iressa(-) Iressa(-) ・Iressa inhibited the phosphorylation of many substrate in HCC827.

  28. Alternative pathway is Src-pathway? A549 HCC827 Src Src EGFR EGFR iressa(+)/iressa(-) iressa(+)/iressa(-)

  29. ＜Kinome Profile modification with metformin ＞　(Dr. Osamu Okitsu) activation １　←　peptide No.　→　５５７ suppression ＜Drug A＞ activation １　←　ペプチドID番号　→　５５７ suppression

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