480 likes | 668 Views
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:
E N D
Tyrosine kinases and MAP kinases in cellular functions Jau-Song Yu Department of Cell and Molecular Biology Chang Gung University
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.
Go Growth factors M 1 hr G2 3-4 hr 4n DNA G1 6-12 hr 2n DNA S 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 Discovery of MAPK *Cell cycle progression ~
Translation Control: an overview www.cellsignal.com
PNAS 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.
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 Gel filtration on Superose 12 Phosphoamino acid analysis 1 M KOH treatment
Nature 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
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 Control cell extracts cell extracts a, undigested MAPK Samples 2-D gel membrane KOH treatment autoradiography
3T3 cells + EGF Cell extracts DEAE-cellulose Phenyl-Superose MAPK assay and WB + EGF - EGF 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 SDS-PAGE cut out eluted trypsin digestion 2D HVE/TLC eluted HPLC on C4 RP column
Biochemistry. 1991 Jan 8;30(1):278-86. • Purification 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.
-GEGAYG- -DLKPSN- Oligonucleotide 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 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.
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 Neuronal-like Muscle-like differentiation differentiation RA DMSO 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. CD45 + PP2A pretreament ERK1 silver stain ERK1 + ATP*/Mg - ERK1
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
A B autophosphorylation Fast Flow S ERK1 Heparin-Sepharose A B A B (A) Q Sepharose Mono S (A) B A
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 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.
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.