Map Kinase
(Map = mitogenactivated 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:
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.)
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Scios: a company centered around
map kinase:
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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_Bro
chure_Oct2004-FINAL-20041108.pdf
Booklet
From
Upstate
Biologicals
On
Map Kinase
Steps in growth factor action…
1.
2.
3.
4.
5.
6.
7.
8.
Growth factor binds to a receptor
located in the cell membrane- this
activates:
Grb2
Sos
Ras
Raf
MEK
Map Kinase
What does map kinase (ERK) do?
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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
Map kinase animation
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http://www.biocreations.com/pages/bioanimations.ht
ml
Map kinase and disease:
http://zygote.swarthmore.edu/cell7.html
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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.
Grb2, Sos
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)
3 Types of Map kinases:
1. c-Jun NH2-terminal kinases (JNKs)- this
phosphorylates Jun (in AP-1)
2. p38 MAPK- regulates cell death (apoptosis)
and inflammatory cytokine expression (may be
important in arthritis)
3. 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
ERK :
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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….
Many have used the Xenopus oocyte
to study insulin and cell division
Xenopus oocyte
(dark animal pole, light
vegetal pole)
ovary
Xenopus laevis
XENOPUS laevis
(similar to human…)
WHITE SPOT
MATURATION
(meiotic cell division
or meiosis)
CLEAVAGE
(mitotic cell
division; mitosis)
How does hormone induce meiotic cell
division? (similar to steps in other cells)
1. Insulin binds a receptor in the plasma membrane
– activating:
2. Map kinase
3. CDK1
4. cell division
Don’t need to know this detail..
White spot
http://carbon.cudenver.edu/~bstith/hormpath.htm
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
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
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See Wiernsperger, 1996; Wiernsperger and Bailey, 1999; Witters, 2001
Advantages of metformin
(Glucophage) (don’t memorize)
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As opposed to other diabetes drugs,
metformin benefits cardiovascular system
since it does NOT promote:
- hypertension
-weight gain
-hypoglycemia
-hyperinsulinaemia
-hyperlipidemia,
-macroangiopathy,
-gluconeogenesis
METFORMIN
But we still don’t know how metformin works…
If we did, we could design better drugs
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
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ELISA assay to detect Active ERK
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http://www.agresearch.co.nz/scied/search/tool
s/Elisa/index_elisa.htm#
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http://www.immunospot.com/elisaanimation.html
ELISA is used to detect active
ERK1/2
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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 phosphoERK1/2
ENZYME
phosphoERK1 or 2 is the “Ag” or antigen
(something that binds antibodies)
Different
ways to
make
the blue
color
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???
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….
1. 10 mM Tris, pH 7.4 – buffer keeps pH proper
2. 100 mM NaCl - correct tonicity for frog cells
3. 1 mM EDTA- binds Ca, Mg to inhibit enzymes
4. 1 mM EGTA– really really binds Ca
5. 1 mM NaF – inhibits phosphatases that remove P
6. 20 mM Na4P2O7 - same as NaF
7. 2 mM Na3VO4 - same as NaF
8. 1% Triton X-100 – detergent removes proteins from
9.
membranes
10.10% glycerol – makes solution “heavy” or thick
11.0.1% to 1.0% SDS - same as triton
12.0.5% deoxycholate– same as triton
13.1 mM PMSF inihbits proteases that destroy Map kinase
14.
-very effective but only stable for a short time
15.Protease inhibitor cocktail – we also add more