Title: Tyrosine kinases and MAP kinases
1Tyrosine kinases and MAP kinases in
cellular functions
Jau-Song Yu
Department of Cell and Molecular Biology
Chang Gung University
2Mitogen Activated Protein Kinase Cascades
3Initial literatures for the finding of MAPK
kinases in mammals
For MAPK/ERKs MAPK purification and
identification 1. Ray LB, Sturgill TW. (1987)
Rapid stimulation by insulin of a
serine/threonine kinase in 3T3-L1 adipocytes that
phosphorylates microtubule-associated protein 2
in vitro. Proc Natl Acad Sci U S A.
84(6)1502-6. 2. Price DJ, Nemenoff RA, Avruch
J. (1989) Purification of a hepatic S6 kinase
from cycloheximide-treated Rats. J Biol Chem.
15264(23)13825-33. 3. Hoshi, M., Nishida, E.,
and Sakai, H. (1988) Activation of a
Ca2-inhibitable protein kinase that
phosphorylates microtubule-associated protein 2
in vitro by growth factors, phorbol esters, and
serum in quiescent cultured human fibroblasts. J.
Biol. Chem. 263, 53965401. Gene cloning for
MAPK family 4. Boulton, T. G., Yancopoulos, G.
D., Gregory, J. S., Slaughter, C., Moomaw, C.,
Hsu, J., and Cobb, M. H. (1990). An
insulin-stimulated protein kinase similar to
yeast kinases involved in cell cycle control.
Science 249, 6467. 5. Boulton, T. G., Nye, S.
H., Robbins, D. J., Ip, N. Y., Radziejewska, E.,
Morgenbesser, S. D., DePinho, R. A., Panayotatos,
N., Cobb, M. H., and Yancopoulos, G. D. (1991).
ERKs A family of protein-serine/threonine
kinases that are activated and tyrosine
phosphorylated in response to insulin and NGF.
Cell 65, 663675.
4For JNK/SAPKs JNK/SAPKs purification and
identification 1. Kyriakis, J. M., and Avruch,
J. (1990). pp54 microtubuleassociated protein 2
kinase. A novel serine/threonine protein kinase
regulated by phosphorylation and stimulated by
poly-L-lysine. J. Biol. Chem. 265,
1735517363. 2. Hibi, M., Lin, A., Smeal, T.,
Minden, A., and Karin, M. (1993) Identification
of an oncoprotein- and UV-responsive protein
kinase that binds and potentiates the c-Jun
activation domain. Genes Dev. 7, 21352148. 3.
Kyriakis, J. M., Banerjee, P., Nikolakaki, E.,
Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J.,
and Woodgett, J. R. (1994) The stress-activated
protein kinase subfamily of c-Jun kinases. Nature
369, 156160. 4. Derijard, B., Hibi, M., Wu,
I.-H., Barrett, T., Su, B., Deng, T., Karin, M.,
and Davis, R. J. (1994) JNK1 A protein kinase
stimulated by UV light and Ha-Ras that binds and
phosphorylates the c-Jun activation domain. Cell
76, 10251037.
5For p38MAPKs p38MAPKs purification and
identification 1. Han, J., Lee, J.-D., Bibbs,
L., and Ulevitch, R. J. (1994). A MAP kinase
targeted by endotoxin and hyperosmolarity in
mammalian cells. Science 265, 808811. 2. Lee,
J. C., Laydon, J. T., Mcdonnell, P. C.,
Gallagher, T. F., Kumar, S., Green, D., McNulty,
D., Blumenthal, M. J., Heys, J. R., and
Landvatter, S. W. (1994). A protein kinase
involved in the regulation of inflammatory
cytokine biosynthesis. Nature 372, 739746. 3.
Rouse, J., Cohen, P., Trigon, S., Morange, M.,
Alonso-Llamazares, A., Zamanillo, D., Hunt, T.,
and Nebreda, A. R. (1994). A novel kinase cascade
triggered by stress and heat shock that
stimulates MAPKAP kinase-2 and phosphorylation of
the small heat shock proteins. Cell 78,
10271037.
6Discovery of MAPK
Cell cycle progression
7Translation Control an overview
www.cellsignal.com
8www.cellsignal.com
9PNAS USA 84, 1502-1506 (1987) Rapid stimulation
by insulin of a serine/threonine kinase in
3T3-L1 adipocytes that phosphorylates
microtubule- associated protein 2 in vitro Ray LB
and Sturgill TW Department of Internal Medicine,
University of Virginia School of Medicine,
Charlottesville 22908, USA.
10(No Transcript)
11PNAS USA 85, 3753-3757 (1988) Insulin-stimulated
microtubule-associated protein kinase Is
phosphorylated on tyrosine and threonine in
vivo Ray LB and Sturgill TW
Cells 1-3 mCi/plate, 2 h 80 nM
insulin, 10 min extracts from 10 plates
DEAE-cellulose (0.5 M NaCl eluted)
Phenyl-Superose (FPLC) SDS-PAGE
12pp40 from phenyl-Superose Gel filtration
on Superose 12
Phosphoamino acid analysis
1 M KOH treatment
13Nature 334, 715-718 (1988) Insulin-stimulated
MAP-2 kinase phosphorylates and Activates
ribosomal protein S6 kinase II Sturgill TW, Ray
LB, Erikson E, and Maller JL
MAPK
autophosphorylation
S6K II
PP2Ac
PP1
Autophosphorylation MAPK
MAPK (-
PP1) ( PP1)
S6 Kinase II PP2A or PP1
NaF (for PP2A) or Inhibitor 2 (for PP1)
ATP/Mg/MAPK
ATP/Mg MAPK 40S protein
ATP/Mg SDS-PAGE
SDS-PAGE
14S6K II PP2A or PP1 inhibitor for
ptase ATP.Mg MAPK purified from resting
or insulin-stimulated cells 40S subunit
ATP/Mg SDS-PAGE
? MAP-2 kinase activity ? S6K II kinase
activity ?S6K II reactivating activity
15 Proc Natl Acad Sci U S A. 1989
Sep86(18)6940-3. Evidence that pp42, a major
tyrosine kinase target protein, is a
mitogen-activated serine/threonine protein
kinase.Rossomando AJ, Payne DM, Weber MJ,
Sturgill TW.
EGF-treated Control cell extracts
cell extracts
a, undigested MAPK
Samples 2-D gel membrane KOH treatment
autoradiography
163T3 cells EGF Cell extracts
DEAE-cellulose Phenyl-Superose MAPK assay and
WB
EGF
- EGF
Western Blot by PY antibody
17Nature. 1990 Feb 15343(6259)651-3. Requirement
for integration of signals from two distinct
phosphorylation pathways for activation of MAP
kinase. Anderson NG, Maller JL, Tonks NK,
Sturgill TW.
1832P-MAPK from EGF-treated 32P-labelled 3T3-L1
cells PP2A or CD45 Phosphoaino acid
analysis
19Proc Natl Acad Sci U S A. 1991 Nov
188(21)9508-12. Autophosphorylation in vitro of
recombinant 42-kilodalton mitogen-activated
protein kinase on tyrosine. Wu J, Rossomando AJ,
Her JH, Del Vecchio R, Weber MJ, Sturgill TW.
20EMBO J. 1991 Apr10(4)885-92. Identification of
the regulatory phosphorylation sites in
pp42/mitogen-activated protein kinase (MAP
kinase)Payne DM, Rossomando AJ, Martino P,
Erickson AK, Her JH, Shabanowitz J, Hunt DF,
Weber MJ, Sturgill TW.
2132P-MAPK SDS-PAGE cut out eluted
trypsin digestion 2D HVE/TLC eluted
HPLC on C4 RP column
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24Biochemistry. 1991 Jan 830(1)278-86. Purificatio
n and properties of extracellular
signal-regulated kinase 1, an insulin-stimulated
microtubule-associated protein 2 kinaseBoulton
TG, Gregory JS, Cobb MH. Department of
Pharmacology, University of Texas Southwestern
Graduate School of Biomedical Sciences, Dallas
75235-9041.
25(No Transcript)
26-GEGAYG- -DLKPSN- Oligonucleotide Primers RT-PCR
ERK1 cDNA
27 Proc Natl Acad Sci U S A 1982
Nov79(22)6792-6 Insulin activates a
tyrosine-specific protein kinase in extracts of
3T3-L1 adipocytes and human placentaPetruzzelli
LM, Ganguly S, Smith CJ, Cobb MH, Rubin CS, Rosen
OM.Insulin activates a tyrosine-specific
cAMP-independent protein kinase when added
directly to detergent extracts of differentiated
3T3-L1 adipocytes and humal placental membranes.
The kinase is also activated by antibody to the
insulin receptor and, to a lesser extent, by
proinsulin. It catalyzes the phosphorylation of
the 92,000-dalton component of the insulin
receptor, histone, and casein in each case,
tyrosine is the principal amino acid modified.
Under the conditions used to activate the kinase,
insulin does not affect the rate of
dephosphorylation of the receptor or of histone.
The insulin-activated kinase is copurified with
the human placental insulin receptor until the
final elution from insulin-Sepharose. It remains
to be established whether the kinase and the
insulin receptor are separate molecules.
28 J Biol Chem 1986 Oct 5261(28)12994-9 An
insulin-stimulated ribosomal protein S6 kinase in
3T3-L1 cells.Cobb MH.A protein kinase that is
stimulated from 2-10-fold by insulin and that
phosphorylates ribosomal protein S6 has been
characterized in 3T3-L1 cells. The detection of
this activity in the 100,000 X g supernatant is
facilitated by the presence of beta-glycerol
phosphate or vanadate in the homogenization
buffer. The activity has been purified 55-fold by
chromatography on DEAE-cellulose and
phosphocellulose. The resulting specific activity
is 584 pmol/min/mg of protein. DEAE-cellulose
chromatography followed by gel filtration on
Ultrogel AcA54 or by glycerol gradient
centrifugation suggests that the protein has a
molecular mass of 60,000-70,000 daltons. Mg2,
and to a lesser extent Mn2, will support
phosphorylation of S6 by the activity. No
proteins tested other than ribosomal protein S6
are phosphorylated. Based on its chromatographic
properties and substrate specificity, the enzyme
appears to be distinct from several other protein
kinases that are known to phosphorylate ribosomal
protein S6 in vitro. The complete
characterization and purification of this enzyme
may be essential to the elucidation of the
mechanism of regulation of S6 phosphorylation by
insulin.
29Science. 1990 Jul 6249(4964)64-7. An
insulin-stimulated protein kinase similar to
yeast kinases involved in cell cycle
control. Boulton TG, Yancopoulos GD, Gregory JS,
Slaughter C, Moomaw C, Hsu J, Cobb MH.
Rat brain cDNA library 1.9 kb 367 a.a. 42,038 Da
Rat tissues and cell lines, highly expressed in
CNS
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31Cell. 1991 May 1765(4)663-75. ERKs a family of
protein-serine/threonine kinases that are
activated and tyrosine phosphorylated in
response to insulin and NGF. Boulton TG, Nye SH,
Robbins DJ, Ip NY, Radziejewska E, Morgenbesser
SD, DePinho RA, Panayotatos N, Cobb MH,
Yancopoulos GD.
Rat brain cDNA library screened by ERK1 probe
with low-normal stringency ERK1-3
32Neuronal specificity of ERK2 and 3 AST
astrocytes, glia cells P19 embryocarcinoma
cell Neuronal-like Muscle-like differentiati
on differentiation
RA
DMSO
RA Retionic acid LANGFR low-affinity NGF
receptor
3332P-labeled cells IP by ERK antibodies
autoradiography
PAA for ERK 1
In (B) Cell extracts 0.5 SDS 1 mM DTT
boiling diluted IP
34Rat 1 HIRc B cells insulin
Rat PC12 cells NGF
IP ERK antibody 691 WB PY 691
(for ERK1)
(for ERK1 2)
(for phospho-Tyr)
35 Proc Natl Acad Sci U S A. 1991 Jul
1588(14)6142-6. Microtubule-associated protein
2 kinases, ERK1 and ERK2, undergo
autophosphorylation on both tyrosine and
threonine residues implications for their
mechanism of activation.Seger R, Ahn NG, Boulton
TG, Yancopoulos GD, Panayotatos N, Radziejewska
E, Ericsson L, Bratlien RL, Cobb MH, Krebs EG.
CD45 PP2A pretreament
ERK1 silver stain
ERK1 ATP/Mg
- ERK1
36Biochem J. 1992 Aug 1285 ( Pt 3)701-5. Renaturat
ion and partial peptide sequencing of
mitogen-activated protein kinase (MAP kinase)
activator from rabbit skeletal muscle.Wu J,
Michel H, Rossomando A, Haystead T, Shabanowitz
J, Hunt DF, Sturgill TW.
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38J Biol Chem 1992 Jul 15267(20)14373-81
Purification and characterization of
mitogen-activated protein kinase activator(s)
from epidermal growth factor-stimulated A431
cells.Seger R, Ahn NG, Posada J, Munar ES,
Jensen AM, Cooper JA, Cobb MH, Krebs
EG.Department of Pharmacology, University of
Washington, Seattle 98195.Two peaks of
mitogen-activated protein (MAP) kinase activator
activity are resolved upon ion exchange
chromatography of cytosolic extracts from
epidermal growth factor-stimulated A431 cells.
Two forms of the activator (1 and 2) have been
purified from these peaks, using chromatography
on Q-Sepharose, heparin-agarose, hydroxylapatite,
ATP-agarose, Sephacryl S-300, Mono S, and Mono Q.
The two preparations each contained one major
protein band with an apparent molecular mass of
46 or 45 kDa, respectively, on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
Evidence identifying the MAP kinase activators as
the 46- and 45-kDa proteins is presented. Using
inactive mutants of MAP kinase as potential
substrates, it was found that each preparation of
MAP kinase activator catalyzes phosphorylation of
the regulatory residues, threonine 188 and
tyrosine 190, of Xenopus MAP kinase. These
results support the concept that the MAP kinase
activators are protein kinases. These MAP kinase
kinases demonstrate an apparent high degree of
specificity toward the native conformation of MAP
kinase, although slow autophosphorylation on
serine, threonine, and tyrosine residues and
phosphorylation of myelin basic protein on serine
and threonine residues is detected as well.
39Proc Natl Acad Sci U S A. 1992 Sep
189(17)8205-9. Purification of a murine
protein-tyrosine/threonine kinase that
phosphorylates and activates the Erk-1 gene
product relationship to the fission yeast byr1
gene product.Crews CM, Erikson RL.
Department of Molecular and Cellular Biology,
Harvard University, Cambridge, Massachusetts
02138, USA
40A B
autophosphorylation
Fast Flow S
ERK1
Heparin-Sepharose
A B A B
(A)
Q Sepharose
Mono S
(A)
B
A
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42Sequential protein kinase reactions controlling
cell growth and differentiation Current Opinion
in Cell Biology 6230-238 (1994) Gary L Johnson
and Richard R Vaillancourt
43 Science 1992 Oct 16258(5081)478-80 The
primary structure of MEK, a protein kinase that
phosphorylates the ERK gene product.Crews CM,
Alessandrini A, Erikson RL.Department of
Cellular and Developmental Biology, Harvard
University, Cambridge, MA 02138.Mitogen-activate
d protein (MAP) kinases, also known as
extracellular signal-regulated kinases (ERKs),
are thought to act at an integration point for
multiple biochemical signals because they are
activated by a wide variety of extracellular
signals, rapidly phosphorylated on threonine and
tyrosine, and highly conserved. A critical
protein kinase lies upstream of MAP kinase and
stimulates the enzymatic activity of MAP kinase.
The structure of this protein kinase, denoted
MEK1, for MAP kinase or ERK kinase, was
elucidated from a complementary DNA sequence and
shown to be a protein of 393 amino acids (43,500
daltons) that is related most closely in size and
sequence to the product encoded by the
Schizosaccharomyces pombe byr1 gene. The MEK gene
was highly expressed in murine brain, and the
product expressed in bacteria phosphorylated the
ERK gene product.
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46 Proc Natl Acad Sci U S A 1993 Dec
190(23)10947-51 Raf-1 forms a stable complex
with Mek1 and activates Mek1 by serine
phosphorylation. Huang W, Alessandrini A, Crews
CM, Erikson RL.Department of Cellular and
Developmental Biology, Harvard University,
Cambridge, MA 02138.Recombinant Mek1 and Raf-1
proteins produced in Sf9 cells undergo a tight
association both in vivo and in vitro, which
apparently does not depend on additional factors
or the kinase activity of Mek1 or Raf-1. The
complex can be disrupted by two polyclonal
antibodies raised against Raf-1 peptides.
Coinfection with Raf-1 activates Mek1 gt 150-fold,
and coinfection with Raf-1 and Mek1 activates
Erk1 approximately 90-fold. The activation of
Mek1 by Raf-1 involves only serine
phosphorylation, which is directly proportional
to the extent of Mek1 activation. Phosphopeptide
maps suggest a single Raf-1 phosphorylation site
on mek1.