Transcript Cell

Tyrosine kinases and MAP kinases
in cellular functions
Jau-Song Yu
Department of Cell and Molecular Biology
Chang Gung University
Mitogen Activated Protein Kinase Cascades
Initial 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. 15;264(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, 5396–5401.
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, 64–67.
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, 663–675.
For 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, 17355–17363.
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, 2135–2148.
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, 156–160.
4. De´rijard, 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, 1025–
1037.
For 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, 808–811.
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, 739–746.
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, 1027–1037.
Discovery of MAPK
Go
*Cell cycle progression ~
Growth factors
M
G2
1 hr
3-4 hr
4n DNA
S
G1
6-12 hr
2n DNA
6-8 hr
2-4n DNA
*Signaling in insulin action ~
Cells
insulin
insulin receptor tyrosine kinase (p-Tyr )
???
???
ribosomal protein S6 kinase (RSK) (p-Ser/Thr )
S6 protein phosphorylation (p-Ser )
Increase of protein synthesis
Translation Control: an overview
www.cellsignal.com
www.cellsignal.com
PNAS USA 84, 1502-1506 (1987)
Rapid stimulation by insulin of a serine/threonine kinase
in 3T3-L1 adipocytes that phosphorylates microtubuleassociated protein 2 in vitro
Ray LB and Sturgill TW
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville 22908, USA.
PNAS 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
pp40 from
phenyl-Superose
Phosphoamino acid analysis
Gel filtration on
Superose 12
1 M KOH treatment
Sturgill TW, Ray LB, Erikson E, and Maller JL
+ MAPK
(- PP1)
+ MAPK
(+ PP1)
S6 Kinase II
+ PP2A or PP1
+ NaF (for PP2A) or Inhibitor 2 (for PP1)
+ ATP/Mg/MAPK
+ 40S protein + ATP*/Mg
SDS-PAGE
+ ATP*/Mg + MAPK
SDS-PAGE
+ PP2Ac
+ MAPK
S6K II
Autophosphorylation
+ PP1
autophosphorylation
Nature 334, 715-718 (1988)
Insulin-stimulated MAP-2 kinase phosphorylates and
Activates ribosomal protein S6 kinase II
S6K II
+ PP2A or PP1
+ inhibitor for p’tase
+ 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
Proc Natl Acad Sci U S A. 1989 Sep;86(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
cell extracts
Control
cell extracts
a, undigested MAPK
Samples 2-D gel membrane
KOH treatment autoradiography
3T3 cells
+ EGF
+ EGF
Cell extracts
DEAE-cellulose
- EGF
Phenyl-Superose
MAPK assay and WB
Western Blot by PY antibody
Nature. 1990 Feb 15;343(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.
32P-MAPK
from EGF-treated 32P-labelled 3T3-L1 cells
+ PP2A or CD45
Phosphoaino acid analysis
Proc Natl Acad Sci U S A. 1991 Nov 1;88(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.
EMBO J. 1991 Apr;10(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.
32P-MAPK
eluted
SDS-PAGE cut out eluted
HPLC on C4 RP column
trypsin digestion
2D HVE/TLC
Biochemistry. 1991 Jan 8;30(1):278-86.
Purification and properties of extracellular signal-regulated
kinase 1, an insulin-stimulated microtubule-associated protein 2
kinase
Boulton TG, Gregory JS, Cobb MH.
Department of Pharmacology, University of Texas Southwestern Graduate School of Biomedical Sciences,
Dallas 75235-9041.
-GEGAYG-DLKPSNOligonucleotide
Primers
RT-PCR
ERK1 cDNA
Proc Natl Acad Sci U S A 1982 Nov;79(22):6792-6
Insulin activates a tyrosine-specific protein kinase in
extracts of 3T3-L1 adipocytes and human placenta
Petruzzelli 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 insulinactivated 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.
J Biol Chem 1986 Oct 5;261(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.
Science. 1990 Jul 6;249(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
Cell. 1991 May 17;65(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
Neuronal specificity
of ERK2 and 3
AST: astrocytes, glia cells
P19: embryocarcinoma cell
RA
Neuronal-like
differentiation
DMSO
Muscle-like
differentiation
RA: Retionic acid
LANGFR: low-affinity NGF
receptor
32P-labeled
cells
IP by ERK antibodies
autoradiography
PAA for ERK 1
In (B): Cell extracts + 0.5% SDS + 1 mM DTT
boiling
diluted
IP
Rat 1 HIRc B cells + insulin
Rat PC12 cells + NGF
IP: ERK antibody 691
WB:
PY
691
(for ERK1)
(for ERK1 & 2)
(for phospho-Tyr)
Proc Natl Acad Sci U S A. 1991 Jul 15;88(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.
Biochem J. 1992 Aug 1;285 ( Pt 3):701-5.
Renaturation 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.
J Biol Chem 1992 Jul 15;267(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.
Proc Natl Acad Sci U S A. 1992 Sep 1;89(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
Fast Flow S
A B
Heparin-Sepharose
(A)
Q Sepharose
B
A
Mono S
(A)
autophosphorylation
ERK1
A
B
A
B
Sequential protein kinase reactions controlling cell growth and differentiation
Current Opinion in Cell Biology 6:230-238 (1994)
Gary L Johnson and Richard R Vaillancourt
Science 1992 Oct 16;258(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-activated protein (MAP) kinases, also known as extracellular signalregulated 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.
Proc Natl Acad Sci U S A 1993 Dec 1;90(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 > 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.