1. Map Kinase (Map = mitogen-activated protein kinases) what is a kinase?
Text pages: ;
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 Oct;183(2): )

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.

5. http://www. upstate. com/img/pdf/MAPK_Brochure_Oct2004-FINAL-20041108
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
Map kinase and disease:
From our med center:

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
(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
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

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 If we did, we could design better drugs
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

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. Different ways to make the blue color
ENZYME
phosphoERK1 or 2 is the “Ag” or antigen
(something that binds antibodies)
Different ways to make the blue color

27. TIME AFTER PROGESTERONE
MAP
KINASE
(ERK)
PHOSPHORY-LATION
&
ACTIVATION
CELL
DIVISION
BEGINS
15 MIN MIN 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