The MAP Kinase Family Extracellular stimuli Classical MAP kinases Atypical MAP kinases MAPKKK MLK1/2/3/7; LZK RAF-1/A/B TAK1; TPL2 c-mos MEKK1-4; DLK ASK1/2; MLTK TAO1/2 ASK1 TAK1 MEKK1-4 MEKK2/3 TPL2??? TAK1??? MAPKK MEK1/2 MKK4/7 MKK3/6 MEK5 ERK3 K (?) HIPK2 (?)??? MAPK ERK1/2 JNK1/2/3 p38α/β/γ/δ ERK5 ERK3/4 NLK ERK7 Effectors
Pleiotropic Functions of MAP Kinases proliferation, angiogenesis embryo growth, Wnt signaling, lung function HSC stroma?? proliferation, survival, senescence inflammation, development, cell cycle neural apoptosis, obesity, T-cell defects
Multiple Roles of ERK1/2 in Cell Proliferation
Activation of the Ras-ERK1/2 Pathway in Cancer From Roberts and Der. Oncogene 2007
Regulation of ERK1/2 MAP Kinases by Mitogens Mitogens Raf MEK1 MEK2 ERK1 ERK2 > 160 cellular targets growth proliferation survival
Activation of MAP Kinases by Phosphorylation
Dephosphorylation and Inactivation of MAP Kinases
Activation of MKPs by MAP Kinases ERK-spec fic binding and activation of MKP-3. (A) GST- ERK2, GST-ERK2 K52A,GST-SAPK (JNK2), GST-SAPK (JNK3), and GST-p38 MAP kinases immobilized on glutathione-sepharose beads (9) were incubated with free His-tagged MKP-3 (10) After extensive washing, binding was assessed by protein immunoblotting Bound MKP-3 was detected with a polyclonal antibody to MKP-3 Binding of antibody to rabbit immunoglobulin G coupled to peroxidase was detected by chemilumines-cence His-tagged MKP-3 (5 ng) was used as a positive control (B) Hydrolysis of p-npp by MKP-3 in the presence of theindicated concentrations of eluted GST-ERK2, catalytically inactive GST-ERK2 K52A, GST-SAPK (JNK2), GST- SAPK (JNK3), or GST-p38 MAP kinase Interaction of MKPs with MAPKs. (A) The docking interaction between MAPKs and MKPs. The docking surface in the MKB domain of MKPs, which can be divided into three modules, binds to the corresponding sites in MAPKs. (B) Activation of MKPs by MAPKs. The dual-specificity phosphatase (DSP) domain in MKPs is inactive without its substrate. Binding of activated MAPKs to the MKB domain induces conformational changes in the DUSP domain, which causes the increase of its catalytic activity.
Regulation of ERK1/2 Pathway by Feedback Loops
Docking Sites of MAP Kinase Substrates Docking sites of substrates and regulators of MAP kinases D box (hydrophobic-basic-hydrophobic) DEF (motif F-X-F-P) Figure 2: Docking of ERK2 to MEK1, MKP3 and MNK1. a, The amino-acid sequences of putative docking sites in ERK2 (Xenopus), MEK1 (Xenopus), MKP3 (rat) and MNK1 (human). The asterisks indicate the charged amino acids presumed to be essential for the docking. b, Lysates of NIH3T3 cells co-transfected with the indicated combinations of SR plasmid DNA encoding wild-type (wt) ERK2 or ERK2 D321N and HA-tagged wild-type MEK1 or mutant MEK1 (K3M or K4,5M) were immunoprecipitated (IP) with anti-ha antibody (bottom gel). Co-immunoprecipitated wild-type ERK2 or ERK2 D321N was detected by immunoblotting (IB) with anti- Xenopus ERK2 antibody (top gel). The expression level of ERK2 in each sample was similar (middle gel). Comparable amounts of MEK1 were immunoprecipitated in each lane (bottom gel). c, Binding capacity of ERK2 D321N for wild-type (wt) and mutant (mut) MKP3. Arg 64 and Arg 65 were replaced by methionines in MKP3 mut. MKP3 was tagged with the Myc epitope. d, The binding capacity of ERK2 D321N for wild-type and mutant MNK1. HA-tagged ERK2 and Myc -tagged MNK1 were used. Arg 405, Arg 406 and Arg 407 were replaced by methionines in MNK1 mut. e, The binding capacity of ERK2 D321N, ERK2 D324N and ERK2 D321,324N for MEK1. f, The binding capacity of wild-type ERK2 or ERK2 S323D (Ser 323 replaced by Asp) for MEK1 or MKK6. Methodology for c f as in b, with immunoprecipitation by the appropriate antibodies.
Common Docking Domain of MAP Kinases The CD domains in the three-dimensional structures of ERK2 and p38. Three-dimensional structures of rat ERK2 and human p38. White arrowheads indicate acidic amino acids of the CD domain (Asp 316 and Asp 319 in rat ERK2, and Asp 313, 315 and 316 in human p38). The CD domain of the MAPK family. Identification of two conserved hydrophobic residues near the CD domain in ERK2. A, sequence alignment of the CD domain sequence among several MAP kinases ranging from budding yeast to human. The numbers mark the residues in the ERK2 sequence. The two conserved acidic residues (Asp316 and Asp319 in ERK2) are in bold type, and the two conserved hydrophobic residues (Tyr314 and Tyr315 in ERK2) are underlined. B, ERK2 structure with Asp316 and Asp319 shown in red and Tyr314 and Tyr315 shown in yellow.
Binding Domain to DEF Motif Distinct from CD Molecular-Surface Representation of Activated ERK2 Docking Domains ERK2 interacts with the D-domain substrate RSK through the CD domain (residues D316 and D319, colored green). The DEF docking domain (residues M197, L198, Y231, L232, L235, and Y261, colored yellow) is necessary for interaction with and transactivation of the Ets family transcription factor Elk-1. Importantly, DEF-domain interactions are also required for the induction and activation of immediate early genes (IEGs) such as Egr1 and c- Fos. The dual phosphorylated activation loop of ERK2, pt183-e-py185, is colored red.
Docking Domain of MAP Kinase Kinases DVD domain (Domain for Versatile Docking) required for activation of MAPKKs by MAPKKKs
Mechanisms Determining Response Specificity to ERK1/2 Signaling Output
MAP Kinase Scaffolds and Specificity MAP kinases are activated by common extracellular stimuli A single MAP kinase can be used in multipler modules role of protein scaffolds Roles of protein scaffolds 1. specificity of modules 2. signal amplification 3. Sub-cellular localization
MAP Kinase Scaffolds in Mammals Fig. 4. Specific activation of ERK1, but not ERK2, after cotransfection with MP1. Duplicate dishes of NIH 3T3 cells were transiently cotransfected with HA-tagged ERK1 or ERK2 and either MP1 or control vector. After 24 hours, cells were deprived of serum for 4 hours and then either left untreated or stimulated with fetal calf serum (FCS, 10%) for 10 min. HA-tagged ERKs were immunoprecipitated and assayed with MBP as substrate, essentially as described (10).
Effect of Scaffold or Kinase Concentration on Cellular Signaling Combinatorial inhibition. Signaling down a scaffolded protein kinase cascade is a question of balance. If there is too little scaffold, signaling will be low (left). At an intermediate concentration of scaffold, signaling will be high (center). Once the concentration of scaffold exceeds that of the kinases it binds, the signaling begins to decrease (right).
Spatio-temporal Organization of MAP Kinase Modules the biological response is dictated by the intensity, duration and localization of MAP kinase activity proliferation vs differentiation proliferation vs arrest in G1/senescence
Spatial Control of MAP Kinase Signaling ERK2 (quiescent) ERK2 (GF 60 min) Regulatory mechanisms of nuclear translocation of ERK1/2. In unstimulated conditions, ERK1/2 is bound to MEK1/2 and localizes in the cytoplasm. Upon stimulation, ERK1/2 dissociates from MEK1/2 and translocates to the nucleus by the use of three distinct mechanisms. I, ERK1/2 dimerizes and is actively transported to the nucleus; II, ERK1/2 passively diffuses into the nucleus; III, ERK1/2 passes through the nuclear pore by directly interacting with the NPC. Then, ERK1/2 is dephosphorylated and actively exported from the nucleus.
PEA-15 Sequesters ERK1/2 in the Cytoplasm
Nuclear MEK1 Induces Cell Transformation
Sustained Activation of ERK1/2 Repress Antiproliferative Genes
Interpretation of the Sustained Activation of ERK1/2 MAP Kinases A working model of how c-fos functions as a sensor for sustained ERK activity. Although both transient and sustained ERK signals stimulate c-fos gene expression (step 1), the transient signal decays before c-fos can be produced and translocated to the nucleus. Sustained ERK activity allows c-fos's Ser 374 and Ser 362 residues to be phosphorylated in ERK- and RSK-dependent manners, respectively (step 2). In addition to stabilizing the protein, these phosphorylations allow ERK to interact with the DEF domain. Recruited ERK then phosphorylates c-fos at Thr 325 and Thr 331, amino-terminal of the DEF domain (step 3). These DEF-domain-directed phosphorylations, as well as other uncharacterized ERK/DEFdomain-associated effects, regulate c-fos function.
Amplitude of ERK1/2 Signal Determines Cellular Response
Cross-talk between PKA and ERK1/2 Pathways Model for signal integration by HePTP. a, In the absence of external stimuli,the 'classical' pathway of MAP-kinase activation (originating at a receptor, R1) through Ras, Raf and Mek has a low basal activity, which is counteracted by HePTP. b, Stimulation of receptors (R2) that couple to adenylate cyclase (AC) turn on the new pathway that involves phosphorylation of HePTP at Ser 23 by PKA, leading to dissociation of Erk from HePTP. This release from inhibition results in MAP-kinase activation and subsequent c-fos induction.
Inactivation of MAP Kinases by Phosphorylation of MAP Kinase Kinases A 3 A Blot: αha MEK1 activity (units) 2.5 2 1.5 1 0.5 MEK1 activity (units) 40 35 30 25 20 15 10 5 0 0 0 1 3 5 15 30 1h 3h 6h exp Time (min) C B 0 1 3 5 15 30 1h 3h 6h exp Time (min) Blot: P -Ser218/222 Blot: MEK1 Luciferase activity (fold increase) 25 20 15 10 5 0
Specific Roles of ERK1 and ERK2 Isoforms? Erk1 -/- Erk2 -/- +/+ +/- -/- Erk2 +/+ Erk2 +/-
Redondant Roles of Erk1 and Erk2 Isoforms? ERK-specific gene silencing unmasks differential roles for ERK1 and ERK2 in cell signaling and proliferation. (a) Schematic representation (top) of the proviral vector form used in shrna-mediated RNA interference. (b) Wild type (+), ERK1 KD or ERK2 KD MEFs were serum starved for 24 h and then stimulated with 20% serum for 5, 10, 30, 60 and 120 min. Western blots were analyzed with anti-phospho-erk and anti-erk antibodies. (c) Bands from (b) were quantified and fold increases in phospho-erk2 or phospho-erk1 levels over total ERK2 or total ERK1 levels calculated. (d) Growth curve of wild-type, ERK1 and ERK2 KD fibroblasts and their corresponding controls, seeded in triplicate in the presence of 10% serum and 2 µg/ml puromycin and counted after the indicated times. Analysis of MEFs KO for Erk1 and Erk2 Cell proliferation (fold) 7 6 5 4 3 2 1 0 WT ERK1 -/- 0 1 2 3 4 5 6 Time (days) * Cell proliferation (fold) 5 4 3 2 1 0 WT ERK2 -/- * * 0 1 2 3 4 5 6 Time (days) Cell proliferation (fold) 5 4 3 2 1 ERK1 +/+ ERK2 f/f ERK1 -/- ERK2 Δ/Δ 0 0 1 2 3 4 5 Time (days)
Cell Proliferation Rate Correlates with the Total Level of Activated ERK1/2
Jnk1 et Jnk2: Effects on c-jun and Proliferation? Results KO MEFs: opposite effects of Jnk1 and Jnk2 Chemical genetic approach: redondant roles of Jnk1 and Jnk2