Reversible Phosphorylation of Proteins

Protein/Enzyme

Protein/Enzyme

OH

PO4

Protein kinaseProtein phosphatase

Cellular Processes:

Metabolism, contractility, membrane transport and secretion,transcription and translation of genes, cell division,fertilization, memory, carcinogenesis, apoptosis, etc.

(Ser, Thr or Tyr)

The 1992 Nobel Prize in Physiology or MedicineNOBELFÖRSAMLINGEN 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".  SummaryThousands 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.

The 2001 Nobel Prize in Physiology or Medicine8 October 2001The 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"

SummaryAll 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.

Strategy for kinase activity detection in cells

Time

Gen

e level

Kin

ase A

Kin

ase B

Kin

ase c

Kinetic profiles of kinase activity and cell function change in cells

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 g) *incubation (1 h, 4oC) *add protein A/G-S4B (50% v/v, 25 l, shaking) *centrifugation (6000 rpm, 1min, 4oC) *wash/cfg 3 times in Buffer BImmunoprecipitates *suspended in 20 l Buffer A *substrate (5-10 g), [-32P]ATP.Mg2+ (0.2-20 mM) *shaking for 10-30 min at RT *adding SDS-sample bufferSDS-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 dithiothreitolBuffer B --- 0.5 M NaCl in buffer A

(quantitative method)

JNK activity assay in IP

Kinase assay by immunoblotting with phospho-specific Ab

(Qualitative to semi-quantitative method)

JNK1

p-JNK1

C CL P 0 0.5 1 1.5 2 3 4

Time post PDT (hr)

p-JNK2

JNK activity assay by Western blot

In-gel kinase assay

Activation of kinases by osmotic shock (In-gel kinase assay)

Elucidation of kinase pathway involved in specific signal events by specific activators and inhibitors

Activation of MAPK by osmotic shock and inhibition of osmotic shock-activated MAPK by PD98059

(In-gel kinase assay)

(-PD98059) (-PD98059) (+PD98059)

Elucidation of kinase pathway involved in specific signal events by anti-sense oligonucleotide against specific kinase

Inhibition of JNK1 by anti-sense oligonucleotide attenuates caspase-3 activation by of photodynamic treatment

Elucidation of kinase pathway involved in specific signal events by specific dominant active/inactive mutants of regulatory proteins

Rac1 mutants:N17 (T>N) -V12 (G>V) +L61 (Q>L) +

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