Map Kinase Map mitogenactivated protein kinases what is a kinase

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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

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Insulin and many growth factors or mitogens act
through Map kinase
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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
• Alzheimers (Exp Neurol. 2003 Oct183(2)394-405.)

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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

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• http//www.upstate.com/img/pdf/MAPK_Brochure_Oct20
  04-FINAL-20041108.pdf

Booklet From Upstate Biologicals On Map Kinase
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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

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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

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Map kinase animation

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

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Growth factors and map kinase Fig. 14-18
Steps missing
Jun is part of AP-1.
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Grb2, Sos
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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)
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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

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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.

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Many have used the Xenopus oocyte to study
insulin and cell division
Xenopus oocyte (dark animal pole, light vegetal
pole)
ovary
Xenopus laevis
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XENOPUS laevis (similar to human)
WHITE SPOT
MATURATION (meiotic cell division or meiosis)
CLEAVAGE (mitotic cell division mitosis)
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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

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Dont need to know this detail..
White spot
http//carbon.cudenver.edu/bstith/hormpath.htm
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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
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• 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

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CLINICAL USES OF METFORMIN(DONT 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

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Advantages of metformin (Glucophage) (dont
memorize)

• As opposed to other diabetes drugs, metformin
  benefits cardiovascular system since it does NOT
  promote
• - hypertension
• -weight gain
• -hypoglycemia
• -hyperinsulinaemia
• -hyperlipidemia,
• -macroangiopathy,
• -gluconeogenesis

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METFORMIN
But we still dont know how metformin works
If we did, we could design better drugs
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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 insulinthat
  would help diabetics

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ELISA assay to detect Active ERK

• http//www.agresearch.co.nz/scied/search/tools/Eli
  sa/index_elisa.htm
• http//www.immunospot.com/elisa-animation.html

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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

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Differentways to make the bluecolor
ENZYME
phosphoERK1 or 2 is the Ag or antigen
(something that binds antibodies)
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MAP KINASE (ERK) PHOSPHORY-LATION ACTIVATION
CELL DIVISION BEGINS
15 MIN 30 MIN 2 TO
3 HRS
TIME AFTER PROGESTERONE
DOREES RESEARCH LAB IN FRANCE FINDS TWO PEAKS OF
MAP KINASE ACTIVAITON JAMES MALLERS LAB HERE AT
UC MED SCHOOL, FINDS ONLY THE LATER
INCREASE--MAYBE MALLERS METHOD IS NOT VERY
SENSITIVE???
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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.
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• 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