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Map Kinase (Map = mitogen-activated protein kinases) what is a kinase?

Map Kinase (Map = mitogen-activated protein kinases) what is a kinase?. Text pages: 406-411; 596-597 Go through web sites on map kinase- see course web page. Insulin and many growth factors or mitogens act through Map kinase. MAPK overstimulation may cause:.

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Map Kinase (Map = mitogen-activated protein kinases) what is a kinase?

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  1. Map Kinase (Map = mitogen-activated protein kinases)what is a kinase? Text pages: 406-411; 596-597 Go through web sites on map kinase- see course web page

  2. Insulin and many growth factors or mitogens act through Map kinase

  3. MAPK overstimulation may cause: • some breast cancers are resistant to standard anti-estrogen therapy and are highly invasive. • chemotherapy-resistant pancreatic cancer • human melanoma cells • Rheumatoid arthritis • Alzheimer’s (Exp Neurol. 2003 Oct;183(2):394-405.)

  4. Scios: a company centered around map kinase: • MAP kinase controls the production of growth factors and inflammatory cytokines, the molecules produced by the immune system that cause inflammation. • Scios believes that inhibition of MAP kinase could reduce the expression of these and other proteins important in the development and progression of inflammatory disease and cancer. • http://www.sciosinc.com/scios/p38

  5. http://www.upstate.com/img/pdf/MAPK_Brochure_Oct2004-FINAL-20041108.pdfhttp://www.upstate.com/img/pdf/MAPK_Brochure_Oct2004-FINAL-20041108.pdf Booklet From Upstate Biologicals On Map Kinase

  6. Steps in growth factor action… • Growth factor binds to a receptor • located in the cell membrane- this activates: • Grb2 • Sos • Ras • Raf • MEK • Map Kinase

  7. What does map kinase (ERK) do? • Map kinase activates CDK1 which turns on cell division (remember cyclin helps…) • Map kinase enters the nucleus and activates transcription factors like AP-1. • Transcription factors then bind to DNA to turn on genes that lead to cell division

  8. Map kinase animation • http://www.biocreations.com/pages/bioanimations.html • Map kinase and disease: http://zygote.swarthmore.edu/cell7.html • From our med center: http://mama.uchsc.edu/vc/cancer/signal/p3.cfm

  9. Growth factors and map kinase Fig. 14-18 Steps missing Jun is part of AP-1.

  10. Grb2, Sos

  11. This is a kinase cascade: Raf turns on MEK by putting phosphates on it, MEK turns on map kinase by putting phosphates on it (end of kinase cascade). Once on, map kinase puts phosphates on transcription factors like Jun, which combine to form AP-1, this turns on AP-1. AP-1 turns on genes for cell division (cyclin, cdk, etc)

  12. 3 Types of Map kinases: • c-Jun NH2-terminal kinases (JNKs)- this phosphorylates Jun (in AP-1) • p38 MAPK- regulates cell death (apoptosis) and inflammatory cytokine expression (may be important in arthritis) • Extracellular signal-related kinases (ERKs)- crucial in cell division, memory and learning (abnormal ERKs may lead to Alzheimers)– WE WILL MEASURE ERK ACTIVATION BY GROWTH FACTORS

  13. ERK : • ERK has two forms: • ERK1 (44kDa) • ERK2 (42kDa) INSULIN (A PROTEIN) OR PROGESTERONE (A STEROID) ADDITION TO XENOPUS OOCYTES INDUCES THE OOCYTE TO UNDERGO (MEIOTIC) CELL DIVISION….

  14. Many have used the Xenopus oocyte to study insulin and cell division Xenopus oocyte (dark animal pole, light vegetal pole) ovary Xenopus laevis

  15. XENOPUS laevis (similar to human…) WHITE SPOT MATURATION (meiotic cell division or meiosis) CLEAVAGE (mitotic cell division; mitosis)

  16. How does hormone induce meiotic cell division? (similar to steps in other cells) • Insulin binds a receptor in the plasma membrane – activating: • Map kinase • CDK1 • cell division

  17. Don’t need to know this detail.. White spot http://carbon.cudenver.edu/~bstith/hormpath.htm

  18. Diabetes results when insulin no longer stimulates the cell…. The antidiabetic drug METFORMIN (trade name Glucophage) fights diabetes; makes insulin more effective, mimics insulin. We found that metformin speeds insulin action in the oocyte…

  19. SINCE THE MIDDLE AGES, Galega officinalis (GOAT RUE, FRENCH LILAC) WAS TAKEN TO RELIEVE SYMPTOMS OF DIABETES. THE ACTIVE INGREDIENT THAT LOWERS BLOOD GLUCOSE IS GUANIDINE. • METFORMIN IS A BIGUANIDE

  20. CLINICAL USES OF METFORMIN(DON’T MEMORIZE!) • Increases survival rate in myocardial infarction and stroke • Lowers blood glucose (predominantly through an increase the translocation of glucose transporters to the cell surface,a stimulation of insulin-mediated muscle glucose uptake and glycogen synthesis) • Increases insulin sensitivity • Inhibits adipose tissue lipolysis, • Reduces circulating free fatty acids • Diminishes hepatic glucose (via gluconeogenesis) • Stimulates insulin receptor tyrosine kinase activity • May PREVENT diabetes in insulin resistant individuals See Wiernsperger, 1996; Wiernsperger and Bailey, 1999; Witters, 2001

  21. Advantages of metformin (Glucophage) (don’t memorize) • As opposed to other diabetes drugs, metformin benefits cardiovascular system since it does NOT promote: - hypertension -weight gain -hypoglycemia -hyperinsulinaemia -hyperlipidemia, -macroangiopathy, -gluconeogenesis

  22. METFORMIN But we still don’t know how metformin works… If we did, we could design better drugs

  23. To study metformin, • We will test whether metformin turns on ERK 1 and 2 with an ELISA • ERK1 and 2 are active when phosphate is put on them (highly negative phosphate makes new weak ionic bonds that tug and pull on the ERK to change its 3-D shape) • Perhaps metformin weakly mimics insulin…that would help diabetics

  24. ELISA assay to detect Active ERK • http://www.agresearch.co.nz/scied/search/tools/Elisa/index_elisa.htm# • http://www.immunospot.com/elisa-animation.html

  25. ELISA is used to detect active ERK1/2 • Note that active ERK1/2 has phosphate on it • So, we want to detect phospho-ERK1/2 • We use an antibody that only binds to ERK1/2 (not to inactive nonphosphorylated ERK1/2) • Then we detect the antibody by attaching an enzyme that makes a blue color • More blue color, more activated phospho-ERK1/2

  26. Differentways to make the bluecolor ENZYME phosphoERK1 or 2 is the “Ag” or antigen (something that binds antibodies)

  27. MAP KINASE (ERK) PHOSPHORY-LATION & ACTIVATION CELL DIVISION BEGINS 15 MIN 30 MIN 2 TO 3 HRS TIME AFTER PROGESTERONE DOREE’S RESEARCH LAB IN FRANCE FINDS TWO PEAKS OF MAP KINASE ACTIVAITON JAMES MALLER’S LAB HERE AT UC MED SCHOOL, FINDS ONLY THE LATER INCREASE--MAYBE MALLER’S METHOD IS NOT VERY SENSITIVE???

  28. We need to get cells, put them in our O-R2 solution, add insulin, wait, and then homogenize the cells and look for phosphorylated map kinase (ERK). We homogenize in a solution that maintains the phosphates on map kinase (ERK).Go through the chemicals in this solution and what they do….

  29. 10 mM Tris, pH 7.4 – buffer keeps pH proper • 100 mM NaCl - correct tonicity for frog cells • 1 mM EDTA- binds Ca, Mg to inhibit enzymes • 1 mM EGTA– really really binds Ca • 1 mM NaF – inhibits phosphatases that remove P • 20 mM Na4P2O7 - same as NaF • 2 mM Na3VO4 - same as NaF • 1% Triton X-100 – detergent removes proteins from • membranes • 10% glycerol – makes solution “heavy” or thick • 0.1% to 1.0% SDS - same as triton • 0.5% deoxycholate– same as triton • 1 mM PMSF inihbits proteases that destroy Map kinase • -very effective but only stable for a short time • Protease inhibitor cocktail – we also add more

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