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Reversible Phosphorylation of Proteins

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Reversible Phosphorylation of Proteins. OH. Protein/Enzyme. Protein phosphatase. Protein kinase. Protein/Enzyme. PO 4. (Ser, Thr or Tyr). Cellular Processes:. Metabolism, contractility, membrane transport and secretion, transcription and translation of genes, cell division,

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slide1

Reversible Phosphorylation of Proteins

OH

Protein/Enzyme

Protein phosphatase

Protein kinase

Protein/Enzyme

PO4

(Ser, Thr or Tyr)

Cellular Processes:

Metabolism, contractility, membrane transport and secretion,

transcription and translation of genes, cell division,

fertilization, memory, carcinogenesis, apoptosis, etc.

slide2

The 1992 Nobel Prize in Physiology or Medicine

NOBELFÖRSAMLINGEN KAROLINSKA INSTITUTETTHE NOBEL ASSEMBLY AT THE KAROLINSKA INSTITUTE (12 October 1992)

The Nobel Assembly at the Karolinska Institute has today decided to award the Nobel Prize in Physiology or Medicine for 1992 jointly to Edmond H. Fischer and Edwin G. Krebs for their discoveries concerning "reversible protein phosphorylation as a biological regulatory mechanism".

Summary

Thousands of proteins participate in a complex interplay in a cell. They are the tools of the living organism, regulating its reactions and activities. For example, proteins maintain the metabolic flux, dictate growth and cellular division, release hormones, and mediate muscular work. Protein interactions are strictly controlled. One of the most important regulatory mechanisms is reversible protein phosphorylation. This means that enzymes phosphorylate and dephosphorylate proteins. Both these enzymatic processes are in turn regulated, often in several steps, allowing amplification and fine control. The 1992 Nobel Prize in Physiology or Medicine is awarded to the American biochemists Edmond Fischer and Edwin Krebs. They purified and characterized the first enzyme of this type. Their fundamental finding initiated a research area which today is one of the most active and wide-ranging. Reversible protein phosphorylation is responsible for regulation of processes as diverse as mobilization of glucose from glycogen, prevention of transplant rejection by cyclosporin, and development of a cancer form like chronic myeloic leukemia.

slide4

The 2001 Nobel Prize in Physiology or Medicine

8 October 2001

The Nobel Assembly at Karolinska Institutet has today decided to award The Nobel Prize in Physiology or Medicine for 2001jointly to Leland H. Hartwell, R. Timothy (Tim) Hunt and Paul M. Nurse for their discoveries of "key regulators of the cell cycle"

Summary

All organisms consist of cells that multiply through cell division. An adult human being has approximately 100 000 billion cells, all originating from a single cell, the fertilized egg cell. In adults there is also an enormous number of continuously dividing cells replacing those dying. Before a cell can divide it has to grow in size, duplicate its chromosomes and separate the chromosomes for exact distribution between the two daughter cells. These different processes are coordinated in the cell cycle. This year\'s Nobel Laureates in Physiology or Medicine have made seminal discoveries concerning the control of the cell cycle. They have identified key molecules that regulate the cell cycle in all eukaryotic organisms, including yeasts, plants, animals and human. These fundamental discoveries have a great impact on all aspects of cell growth. Defects in cell cycle control may lead to the type of chromosome alterations seen in cancer cells. This may in the long term open new possibilities for cancer treatment.

slide8

Kinetic profiles of kinase activity and cell function change in cells

Gene level

Kinase A

Kinase B

Kinase c

Time

slide9

Kinase assay in immunoprecipitate (IP)

Cells

*homogenization (10-cm dish/0.5 ml lysis buffer)

*centrifugation (12000~15000 rpm, 15 min, 4oC)

Supernatants

*protein concentration determination

*1 mg protein/0.5 ml extracts

*add Ab against specific kinase (5 mg)

*incubation (1 h, 4oC)

*add protein A/G-S4B

(50% v/v, 25 ml, shaking)

*centrifugation (6000 rpm, 1min, 4oC)

*wash/cfg 3 times in Buffer B

Immunoprecipitates

*suspended in 20 ml Buffer A

*substrate (5-10 mg), [g-32P]ATP.Mg2+ (0.2-20 mM)

*shaking for 10-30 min at RT

*adding SDS-sample buffer

SDS-PAGE

Autoradiograpgy

Lysis buffer-----10 mM Tris-HCl at pH 7.4, 2 mM EDTA, 1 mM EGTA,

1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride,

0.5 mg/ml aprotinin

Buffer A --- 20 mM Tris-HCl at pH 7.0, 0.5 mM dithiothreitol

Buffer B --- 0.5 M NaCl in buffer A

(quantitative method)

slide11

Kinase assay by immunoblotting with

phospho-specific Ab

(Qualitative to semi-quantitative method)

slide12

Time post PDT (hr)

C CL P 0 0.5 1 1.5 2 3 4

p-JNK2

p-JNK1

JNK1

JNK activity assay by Western blot

slide15

Elucidation of kinase pathway involved in specific

signal events by specific activators and inhibitors

slide17

Activation of MAPK by osmotic shock and inhibition

of osmotic shock-activated MAPK by PD98059

(+PD98059)

(-PD98059)

(-PD98059)

(In-gel kinase assay)

slide19

Elucidation of kinase pathway involved in specific signal

events by anti-sense oligonucleotide against specific kinase

slide20

Inhibition of JNK1 by anti-sense oligonucleotide attenuates

caspase-3 activation by of photodynamic treatment

slide21

Elucidation of kinase pathway involved in specific

signal events by specific dominant active/inactive

mutants of regulatory proteins

slide22

Rac1 mutants:

N17 (T>N) -

V12 (G>V) +

L61 (Q>L) +

slide24

Gleevec (STI571)

Inhibitor of c-Abl

for CML treatment

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