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Mitogen-Activated Protein Kinase 1

Mitogen-Activated Protein Kinase 1. Emerlyn Goh. Overview. Introduction Structure Function Related Molecules Specific Aspect * References. Introduction. What is a kinase? Phosphorylation What are mitogen-activated protein kinases?

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Mitogen-Activated Protein Kinase 1

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  1. Mitogen-Activated Protein Kinase 1 Emerlyn Goh

  2. Overview • Introduction • Structure • Function • Related Molecules • Specific Aspect* • References

  3. Introduction • What is a kinase? • Phosphorylation • What are mitogen-activated protein kinases? • a.k.a MAPKs (microtubule-associated protein kinase/mitogen activated protein kinase), ERKs (extracellular signal-regulated kinases) • History • Signal transduction pathways • Highly Conserved • Transmembrane communication • MAP2K, MAP3K

  4. MAP Kinase Cascades Source: CALBIOCHEM et al, 2008

  5. ERK1/2 Signal Transduction Pathway • ERK1/2 similar (ERK2 focus) • ERK2 = MAPK1 • Mitogen(EGF)Receptor (RTK)GTPase(ras)Protein kinase (raf)MAPK cascadecellular response • Often called “Ras-Raf-MEK-ERK” pathway • Mechanisms of activation of MAP3K largely unknown • Thr-575 important

  6. The MAPK Signal Transduction Pathway(Krysan)

  7. MAP2K (MEK1) ActivationPDB ID 1s9j (Ohren et al, 2004)

  8. Structure of ERK2 • Inactive form: Tyr-185 blocks active site • 2 domains further apart • Active form: Tyr-195, Thr-183 • Dual phosphorylation • Alignment of p-tyrosine & p-threonine, turn towards surface arginine-rich binding sites to maximize ATP binding • Local and global conformation changes • Active site closure

  9. Unphosphorylated ERK2 Phosphorylated ERK2 PDB ID 1ERK (Zhang et al, 1997) and 2ERK (Canagarajah et al, 1998)

  10. Structure Comparison: Activated/Inactivated Residues PDB ID 1ERK (Zhang et al, 1997) and 2ERK (Canagarajah et al, 1998)

  11. Functions of ERK2 • Regulates proliferation and differentiation of post-mitotic cells • Development of the mesoderm/placenta in many eukaryotes* • Cell growth (ex. transcription factor Elk-1) • Phosphorylation of P53 in cervical/breast carcinoma cells by ERK2 suggests apoptosis • Cell cycle progression and cancer expression (ex. transcription factor c-myc)

  12. Functions, Explained • Elk-1 : transcription factor involved in the expression of c-fos (Gille et al, 1995) • C-fos accounts for cell differentiation, growth • P53 : Breast carcinoma cells + doxorubicin  activation of ERK2  phosphorylates P53 on Thr-55 residue  suggests apoptosis in some cells and drug resistance in cancerous mutants (Pei et al, 2004) • C-Myc : proto-oncogene regulated by MAPK pathway • Overexpression pancreatic/colon cancers • Cyclin expression and G1-S progression • Cancer treatment? (Marampon et al, 2006)

  13. Related Molecule: MKP3 • Dual specificity MAPK phosphatases (MKPs/DSPs) • MKP3 (DUSP6) prototypical ERK2-specific • Able to form stable complex with ERK2 • KIM peptide binds to N-terminal noncatalytic site opposite activation loop of ERK2 • Tightly bound for catalytic activation, dephosphorylation of ERK2 • Underexpression  ERK2 overexpressionpancreatic carcinogenesis

  14. MKP3 KIM PeptideComplexed with ERK2PDB ID 1fys (Liu et al, 2006)

  15. Activation of MKP3 • Activation site is a shallow cleft to accommodate Tyr/Thr residues on ERK2 • Inactivated MKP3: Asp-262 found in loop 5.5Å away from active site • Once ERK2 binds: Asp-262 loop folds to bring aspartate into active site catalytically active stable conformation (Theodosiou et al, 2002)

  16. Unactivated MKP3(PDB ID 1MKP Stewart et al, 1998) PDB ID 1MKP (Stewart et al, 1999)

  17. How is MKP3 Regulated? • Posttranslational mechanism by ERK2 • Via MEK-ERK pathway MKP3 phosphorylated on Ser-159, Ser-197 • Phosphorylation on either serine enhances proteasomal degradation of MKP3 • Experiment: Double mutations on the serines caused 3-fold increase in MKP3 half-life • This is a positive feedback loop by ERK2 by promoting degradation of one of its main inhibitors (Marchetti et al, 2005)

  18. Related Molecule: Aurora-A Kinase • AURKA gene • Key roles in cell mitosis • Potential oncogene, uncertain • Pancreatic cancer linked to hyperactivation and overexpression of Aurora-A by MAPK pathway • Drug treatment options? • (Fu et al, 2007)

  19. Aurora-A Phosphorylated/Unphosphorylated Compared • Unactive Thr exposed for phosphorylation • Phosphorylated Thr pulls inward to a buried positionactive conformation PDB ID 1OL7 Bayliss et al, 2003 and PDB ID 1MQ4 Nowakowski et al, 2003

  20. Essential Role of ERK2 in Mesodermal and Placental Development • Experiment: ERK2-deficient mice embryos  placental development problems • ERK2-deficient mice poor fetal vascularization in placentasevere defect in labyrinthine layer of placentapoor development • As a result fetuses exhibited extreme growth retardation, thin heart walls, and death (Hatano et al, 2003) • Experiment: ERK2 mutant embryos failed to develop mesoderm, defective differentiation (Li et al, 2003)

  21. Codon 99 Mutant of ERK2 Wildtype, Heterozygous, and Homozygous compared-Arrowhead=fetal nucleated erythrocytes-Arrows=mother nucleated erythrocytes-Heterozygoushypomorphic alleleHatano et. al, 2003

  22. References • Gille et al, 1995: http://www.ncbi.nlm.nih.gov/pubmed/8548291 • Pei et al, 2004: http://www.nature.com/onc/journal/v23/n20/full/1207426a.html#bib41 • Waas et al, 2007: http://www.ncbi.nlm.nih.gov/pubmed/17251036?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum • Theodosiou et al, 2002: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=139386 • Marchetti et al, 2005: http://www.ncbi.nlm.nih.gov/pubmed/15632084 • Fu et al, 2007: http://mcr.aacrjournals.org/cgi/content/full/5/1/1?ck=nck • Hatano et al, 2003: http://www.genestocellsonline.org/cgi/reprint/8/11/847.pdf • Stewart et al, 1998: PDB ID 1MKP Structure of MKP3 • Liu et al, 2006: PDB ID 1FYS • Ohren et al, 2004: PDB ID 1s9j • Krysan, http://www.hort.wisc.edu/Krysan/) • Zhang et al, 1997: PDB ID 1ERK • Canagarajah et al, 1998: PDB ID 2ERK • Calbiochem et al, 2008: http://www.emdbiosciences.com/html/CBC/phosphorylation_inhibitors_mitogen-activated_protein_kinase.htm • Li et al, 2003: http://www.pnas.org/cgi/content/abstract/100/22/12759 • Marampon et al, 2006: http://www.molecular-cancer.com/content/5/1/31

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