1. Protein phosphorylation Biochemistry & diversity MAPKs PKB Take-home lessons –Conformational changes –Phosphorylation-dependent kinases –Specificity through complex formation

2. Kinase architecture Kinase domain Regulatory domain –Pseudosubstrate –Allosteric –External subunit Targeting domain –Phospho-tyrosine binding (SH2/SH3) –Phospholipid binding (PH, C2) –Protein

3. Kinase diversity Serine/Threonine –AGC kinases – allosteric regulation –CaMK – CaM regulation –MAP kinases – regulation by phosphorylation Tyrosine –RTKs – Ligand regulation –Non-receptor tyrosine kinases Cofactor PH cascade

4. Phosphorylation PO 4 - group charge alters the electron balance of power Phosphorylatable residues –Polar OH group Serine ThreonineTyrosine N CC OH H CH 2 N CC OH H N CC OH H OH CHCH 2 CH 3 OH

5. Conformational change ~45 kJ from ATP hydrolysis ~10 kJ per ionic bond ~5 kJ per H-bond

6. Base substitution Experimentally modify residues to mimic phosphorylated/non-phosphorylated form Serine N CC OH H CH 2 OH Phosphoserine N CC OH H CH 2 OPO 3 - N CC OH H CH 3 Alanine N CC OH H CH 2 C OO-O- Glutamic Acid

7. Experimental Manipulation Residue substitution in 4EBP1 Growth assay Immunoprecipitation -riiydrkflmecrnspvtkt- UCA ACC Wild type mRNA: Wild type protein: -riiydrkflmecrnApvAkt- Neutral=binds eIF4E GCC GCA Modified protein: Modified mRNA: -riiydrkflmecrnEpvEkt- Acidic=releases eIF4E GAG Modified protein: Modified mRNA:

8. MAP kinases Ubiquitous S/T kinases –Common effectors for many signals Diverse family –ERK –SAPK (JNK) –p38 Cascade –MAPK –MAPKK (MEK) –MAPKKK (MEKK)

9. MAP kinases Activated by extracellular stimulus –Mitogen –Chemical stressor –Physical stressor Receptor mediated –Adapter protein

10. MAPK common structure Nucleotide binding pocket Substrate docking site –Docking domain –Separate substrate domain Docking domain Active site ATP pocket p38 MAPK Callaway et al 2005

11. MAPK subfamilies Extracellular signal regulated kinase ERK –Raf-MEK 1/2-ERK ½ –Growth factor stimulated c-Jun N-Terminal Kinase JNK (SAPK) –MEKK 1-4 – MKK 4/7 – JNK 1/2/3 –Stress (UV light, oxidative, heat) stimulated p38 –TAK/ASK – MKK 3/6 – p38  –Stress/cytokine stimulated

12. MAPK families Raf MEK 1/2 ERK 1/2 GF Elk1,AP-1, MNK, RSK, PLA2 MEKK1/2/3/4 MKK 4/7 JNK 1/2 Cytokines AP-1, NFAT, IRS-1, HSP, BIM/Bcl2 MEKK1/2/3/4, TAK MKK 3/6 P38, SAPK Stress, cytokines CREB, SRF, MEF2, MNK, HSP, PLA2 MAPKKK MAPKK MAPK Stimulus Effectors

13. ERK 1/2 Widman et al. 1999

14. ERK 1/2 Transcription factor activation –Elk1, cMyc, cFos, cJun (C-term), AP-1 –Immediate/early gene upregulation Cell proliferation –cdk2, stimulate G1-S transition –G2-M transition Cellular function –Protein synthesis-MNK –Cytoskeletal remodeling-MAPK-APK

15. eg: Myoblast proliferation Muscle growth/hypertrophy requires satellite cells –Adult, muscle-resident stem cells –Activate, proliferate, and differentiate to allow growth Satellite Cell Myonucleus Basal Lamina Cell membrane Proliferate Differentiate & fuse

16. ERK1/2 control of proliferation Myoblasts are dependent of FGF for growth FGF required for S-phase entry (Clegg et al., 1987) Grow cells in FGF-rich media Count cells in S-phase by nucleotide incorporation Remove FGF at t=0 Keep a few in FGF, just to be sure Without FGF, cells finish their S-phase and don’t start another

17. FGF-mediated ERK signaling FGF  ERK  c-Myc  Cyclins A,B,E; CDK2 c-Myc inhibitor (MadMyc) forces cell cycle withdrawal (Marampon et al., 2006) FGF-ERK represses myogenin expression (Tortorella, 2001) Myc Inhibitor Reduces cell # Increases differentiation Inhibit ERK Increase myogenin

18. JNK Cell stress response –Heat shock –DNA damage –Reactive oxygen –UV light Cytokines –Tumor Necrosis Factor (TNF) receptor –Receptor tyrosine kinase –GPCR

19. JNK Transcription factors –cJun (N-term), NFAT, ATF-2, AP-1, forkhead Receptor modification –IRS-1 inhibition Cellular function –Apoptosis- BCL2, FasL Immune cell activation, cell motility

20. P38 Stress response –Heat, oxidative, UV –Osmotic shock –Cytokines Transcription factors –MEF2, Elk-1, cFos, cJun, CREB Cellular function –Apoptosis/hypertrophy –HSP27, MNK-1

21. Crosstalk MEKK –Most can be activated by RTK, Gq, Gi, PKC… –Substrate preference MEK –Most can be phosphorylated by any MEKK –Substrate specificity varies with isoform MAPK –Common substrates (Elk-1, cMyc, cFos, cJun) –Unique substrates (Cellular, NFAT, HSP)

22. Regulatory processes Scaffold/complex formation provides specificity MAPK negative feedback –Upstream inactivation –Downstream phosphatases Transport and localization

23. MAPK complexes ERK –HSP90 (90 kD heat shock protein) –14-3-3 JNK –IKAP (inhibitor of kappa-B complex associated protein) –JIP (JNK interacting peptide)

24. Scaffolding MAPK (ERK) –Caveolin –14-3-3 –KSR (kinase suppressor of ras) JNK –JIP –Ste5p Assembly Inhibition

25. Scaffolding Classical Cascade Isoform Specification Activation Specification

26. Scaffold differentiation of MAPK MAPKs are mass produced cogs in complex molecular machines Function derives from complex more than cog Elion 2001 Multiple, independent Ste20 dependent behaviors in yeast

27. Negative feedback MKP –MAP kinase phosphatase –Inducible, nuclear/constitutive, cytoplasmic DSP-dual specificity phosphatase

28. Transport and localization Nuclear import/export –Rho/rac –Nuclear scaffolds/nuclear anchoring proteins Cytoplasmic anchor –Some b-arrestin/GPCR complexes – MAPK dimers Subcellular localization –JIP scaffold with kinesin –JNK transport to growth cone

29. PI-3K-akt/PKB cascade Phosphatidylinositol 3’ kinase Phosphoinositide dependent kinase –PIP3 mediated membrane recruitment Protein kinase B (akt) –PIP3 mediated membrane recruitment –PDK1-dependent phosphorylation Mammalian Target of Rapamycin –Translational effectors –Signal integrator – GSK3, TSC, AMPK PI3K PDK1 akt mTOR p70S6k 4EBPeIF2 Receptor

30. mTOR KEGG database http://www.genome.jp