Calcium Signaling
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Hypertrophic signalling • Identify contraction-induced growth signals • Describe the composition and regulation of mTORC1 • Describe the effectors of mTOR • Explain the role of mTOR in muscle hypertrophy – Muscle contraction – Diet – Growth factors Consequences of contraction • Intracellular calcium increase • ATP (energy) turnover – Muscle: Oxygen depletion, AMP accumulation – Systemic: nutrient mobilization • Membrane permeability • Growth factor release – Peptides: IGF-1, FGF, HGF – Lipids: PGF2a, PGE2 • Systemic hormones – Insulin, GH, adrenaline Exercise induces mTOR activity Akt phosphorylation Rats trained to lift 60%BW vest Phosphorylation by WB Protein synthesis over 16 h Rapamycin blocks mTOR phosphorylation • • • • Kubica & al., 2005 Bolster & al., 2003 Rapamycin blocks hypertrophy • Synergist ablation – Cyclosporin to block Cn – Rapamycin to block mTOR • CsA muscles hypertrophy • Rap muscles don’t Bodine & al., 2001 Why mTOR? • Powerful, multiplex regulator of protein synthesis and growth – Translation efficiency – Translational regulation/selection – Protein degradation • Activated by diverse growth and function relevant stimuli – Contraction/exercise – Nutrients – Hormones (insulin, IGF, HGH) Mammalian Target of Rapamycin Pro-growth stimuli mTORC Contraction mTOR p38 Protein synthesis (hypertrophy) Deldicque & al., 2005 Two mTOR Complexes Rapamycin sensitive • mTORC1 Composition – – – – mTOR GbL (mLST8) dispensible PRAS40 RAPTOR • Regulation – Growth factors (PI3K/akt) – Nutrients (TSC1/2, Rag) – Redox • Targets – Ribosomal biogenesis (p70S6k) – Translation (4EBP1) – Autophagy Rapamycin insensitive • mTORC2 Composition – – – – mTOR GbL (mLST8) PRR5, mSin1 RICTOR • Regulation – Growth factors (PI3K/akt) – mTORC1 (RICTOR) • Targets – – – – Cytoskeleton (esp yeast) Proteasome (AktFOXO) Glycogen synthesis (GSK3) PKC Core mTORC1 control • Active complex requires Rheb-GTP – Rheb GTPase – GTPase-Activating Protein (GAP) – Guanine Exchange Factor (GEF) – mTOR autophos S2481 TSC1 • TSC 1/2 – Tuberous Sclerosis Complex – Major site of GF/energy reg. Rheb-GDP TSC2 TCTP(?) Rheb-GTP GbL mTOR RAPTOR • GEF unknown/unnecessary – Translationally Controlled Tumor Protein Substrate Growth Factors and “Energy” • Phosphatidylinositol 3’ kinase (PI3K) – PIP2PIP3 – PDK1 – Akt • • • • • Extracellular-signal Regulated Kinase (ERK) P38MK2 ERK2 MK2 Akt AMPK (activates TSC2) GSK3 TSC1 GSK3 (activates TSC2) TSC2 AMPK Hypoxia Rheb-GTP Rheb-GDP – HIFREDD REDD Amino Acids • Branched-chain AA – Leucine, isoleucine, valine – Rag-GTPase – Ragulator AA-sensitive GEF – Translocation to Rheb-rich lysosomes Rag-GDP TSC2 Ragulator RagB-GTP GbL RAPTOR mTOR Rab7/ lysosome Rheb-GTP Sanack & al., 2008 AA-starved mTOR is distributed through the cytoplasm, and becomes localized to lysosomes rapidly on AA feeding Growth factors and overload • Insulin – Suppressed at low (<60% VO2max) intensity – Neutral at high (>80% VO2max) • Insulin-like growth factor-1 – Elevated after resistance exercise (up to 2 days) – Powerful growth stimulator • Insulin and IGF-1 Receptors – Insulin receptor substrate 1 (IRS1) – PI3KAkt – ERK, p38, PLC IGF-1 expression after synergist ablation (Adams & al 2002) IGF-1 promotes muscle growth • Infused into muscle (not systemic) – Activation of Akt, mTOR – p70S6k, 4EBP1 Adams & McCue 1998 Overload seems independent of IGF-1 • Muscle hypertrophy by synergist ablation in IGF-1R knockout • Cardiac hypertrophy by swim-training in p70S6k knockout WT MKR-/35 d 7d 0d Plantaris mass after synergist ablation Spangenburg & al 2008 Heart weights after 8 weeks swimming (McMullen & al., 2004) Amino acid feeding • AA feeding alone increases mTOR &PS • Protein feeding with exercise gives much better/faster mTOR activation • No difference in hypertrophy (22 weeks) mTOR phosphorylation post-exercise with or without protein feeding (Hulmi & al 2009) Metabolic effects • Elevated AMP – AMP Kinase TSC2 --| mTOR – Permissive? • GSK3 – InsulinAkt--|GSK3 • Oxygen – Hypoxia Inducible Factor REDDTSC2 – ROS directly oxidize cysteines AICAR-induced activation of AMPK blocks AA-induced protein synthesis (Pruznak & al., 2008) Intermediate summary • Exercise-related stresses tend to block mTOR during exercise and activate mTOR after exercise – Energetic stresses during exercise: Low O2, high AMP – Recovery processes/hormones after exercise • Nutrient mobilization • Insulin • IGF-1 • Acute mTOR signaling correlates with hypertrophy under normal conditions – Not in Insulin/IGF-1 receptor defective models – Not in p70 S6k defective models Correlation and causation Type II fiber area 8000 6000 4000 2000 0 Muscle mass gain after 6 weeks HFES correlates with p70S6k phosphorylation at 6 hours. (Baar & Esser 1999) Placebo Protein 5 10 15 Fold phosphorylation of p70S6k 20 Fiber size after 3 weeks training vs p70S6k phosphorylation. (Hulmi & al 2009) mTOR effectors • Ribosome assembly – p70S6kRPS6 – 5’-TOP mRNAs (ribosome components) • Translational efficiency – 4EBP--|eIF4E – Cap dependent translation • Transcription factors – Akt/SGK--|FOXO – NFAT3, STAT3 • IRS-1 (negative feedback) Protein translation • Initiation – eIF4 recognition and melting of 7’mG cap • eIF4E cap-binding subunit • 4EBP competition with eIF4F scaffold – Recruit 40S ribosome • met-tRNA • eIF2 GTP-dependent met-tRNA loader – Recruit 60S ribosome • Start codon Initiation Pre-initiation complex Fig 17-9 Transition to elongation Protein translation • Elongation – tRNA recruitment • eEF1 GTP-dependent tRNA carrier • GTP hydrolysis with peptide bond formation – Ribosome advance • eEF2 GTP-dependent procession • GTP hydrolysis with advance Elongation eEF1 Cycle Elongation Cycle eEF2 cycle Fig 17-10 3’ untranslated region structure • Post-transcriptional control – 2° and 3° structure of mRNA – Analogous to DNA promoter • 5’ Tract of Oligopyrimidines – Ribosomal proteins – eEF1, eEF2 • “Highly structured” 5’ cap – Ribosome scanning – Growth factors, cell cycle control • Internal Ribosome Entry Site (IRES) – Inflammation, angiogenesis Phosphorylated RPS6 favors these Active eIF4 complex favors these Species differences • • • • • Most proteins conserved yeast-human Regulatory processes differ S cerevisiae have 2 TORs Drosophila akt doesn’t directly regulate TSC2 C Elegans has no TSC1/2; transcriptional repression of RAPTOR via FOXO • S cerevisiae mTOR independent of Rheb Summary • High force contractions induce multiple signaling modes – Metabolites, growth factors, mechanical • Hypertrophy closely linked with mTOR – GF signaling – Metabolite signaling • mTOR is a powerful control of protein accretion – Makes more ribosomes via p70S6k – General translation efficiency via 4EBP – Reduced degradation via FOXO, NFAT3
