Reversible Phosphorylation of Proteins

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Reversible Phosphorylation of Proteins
OH
Protein/Enzyme
Protein kinase
Protein phosphatase
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.
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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.
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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.
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Strategy for kinase activity detection in cells
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Kinetic profiles of kinase activity and cell
function change in cells
Gene level
Kinase A
Kinase B
Kinase c
Time
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Kinase assay in immunoprecipitate (IP)
Cells homogenization (10-cm dish/0.5 ml
lysis buffer) centrifugation (1200015000
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-32PATP.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)
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JNK activity assay in IP
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Kinase assay by immunoblotting with
phospho-specific Ab
(Qualitative to semi-quantitative method)
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JNK activity assay by Western blot
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In-gel kinase assay
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Activation of kinases by osmotic shock
(In-gel kinase assay)
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Elucidation of kinase pathway involved in
specific signal events by specific activators
and inhibitors
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Activation of MAPK by osmotic shock and
inhibition of osmotic shock-activated MAPK
by PD98059
(PD98059)
(-PD98059)
(-PD98059)
(In-gel kinase assay)
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Elucidation of kinase pathway involved in
specific signal events by anti-sense
oligonucleotide against specific kinase
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Inhibition of JNK1 by anti-sense oligonucleotide
attenuates caspase-3 activation by of
photodynamic treatment
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Elucidation of kinase pathway involved in
specific signal events by specific dominant
active/inactive mutants of regulatory proteins
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Rac1 mutants N17 (TgtN) - V12 (GgtV) L61 (QgtL)

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NATURE REVIEWS DRUG DISCOVERY VOLUME 1 APRIL
2002 309
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Gleevec (STI571) Inhibitor of c-Abl for CML
treatment
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