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CHMI 4237 E Special topics in Biochemistry Cell proliferation 4- Signaling to the cell cycle – TGF-b Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry Laurentian University CHMI 4237 E - Winter 2010 1 So, what are the BIG questions: 1) How does the basic cell cycle machinery work? 2) How does the cell ensure that a given step in the cell cycle is properly completed before moving forward? 3) What are the signals that modulate the cell cycle? CHMI 4237 E - Winter 2010 2 Transforming growth factor beta Isolated as a component of « sarcoma growth factor »; Triggers a number of biological effects, including cell proliferation and cell cycle arrest; + EGF SGF + PDGF CHMI 4237 E - Winter 2010 M.B. Sporn / Cytokine & Growth Factor Reviews 17 (2006) 3–7 3 Transforming growth factor beta Family of over 33 proteins, which includes: ◦ TGFb ◦ Bone morphogenetic proteins (BMPs) ◦ Activins ◦ Growth and differentiation factors (GDFs) nature cell biology volume 9 | number 9 | SEPTEMBER 2007 Number of effects: ◦ Proliferation (stimulation/inhibtion) ◦ Differentiation ◦ Cell adhesion ◦ Cell migration ◦ Cell death CHMI 4237 E - Winter 2010 4 TGF-b secretion TGF-b is first synthesized on the ribosome as a pre-proprotein; The pre-sequence is removed during insertion into the ER lumen During its transit in the secretory pathway, TGF-b is processed and converted into its secreted form, associated with LTBP; Active TGF is release by the action of a number of factors, including: ◦ Metalloproteases MMP-2 / MMP9 ◦ Plasmin ◦ Integrins (i.e. extracellular matrix) http://www.comparative-hepatology.com/content/figures/1476-5926-6-7-6.jpg CHMI 4237 E - Winter 2010 5 TGF-b receptor TbR-II TGF-b triggers its effects on the cell by causing the dimerization of two subunits of the TGF receptor: Single-span membrane proteins Possess Ser/Thr kinase activity TbR-I subunit: TbR-I ◦ possess a 30-amino acid GS domain preceding the kinase domain TbR-II subunit: Activates receptor in a ligand-specific manner by phosphorylating the GS sequence of TbR-I Doesn’t have a GS sequence; http://jkweb.berkeley.edu/external/pdb/2001/tgf _beta_R1/receptor_schematic.jpg CHMI 4237 E - Winter 2010 6 TbR-I activation In the absence of ligand: TbR-I is inhibited by its GS sequence, which is wedged in the N lobe of the Ser/Thr kinase domain; This prevents ATP binding by the N-lobe; TbR-I is stabilized in this form through the binding of FKBP12; CHMI 4237 E - Winter 2010 http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg 7 TbR-I activation TGF binding causes the dimerization of TbR-I and TbR-II; TbR-II phosphorylates the GS sequence; This is sufficient to dislodge the GS sequence from the N- lobe and allow ATP binding; Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010 8 TbR-I activation Phosphorylated TbR-I acts as a docking site for the actual signal transducers: a family of proteins called R-SMADS; SMADS are brought to the TbR-I/TbR-II dimer by a membranebound protein called SARA; R-SMAD phosphorylation by TbR-I triggers the signaling cascade. CHMI 4237 E - Winter 2010 http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg 9 SMADS Three classes are recognized: ◦ R-SMAD: initiate signaling at the TbR; ◦ SMAD4: modulates the expression of target genes ◦ Inhibitory SMADs: involved in signal termination; Main protein regions: 1) MH1: ◦ Binds DNA at the SMAD binding element (SBE) in the promoter of target genes ◦ Binds a number of transcription factors Signal Transduction. 2nd edition. 2009. Academic Press 2) ◦ ◦ ◦ linker region: hot spot for phosphorylation PPxY motif: binding site for E3 ubiquitin ligase Nuclear export signal (SMAD4 only). 3) MH2: ◦ hydrophobic corridor (patch of hydrophobic amino acids) mediating protein interactions with SARA (cytoplasmic retention), nuclear pore proteins and transcription factors; ◦ SxS motif: phosphorylated by TbR-I CHMI 4237 E - Winter 2010 10 SMADS http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf CHMI 4237 E - Winter 2010 11 SMADS Signal Transduction. 2nd edition. 2009. Academic Press When phosphorylated by TbR-I, the SxS motif interacts with a basic pocket in MH2; This promotes heteromerization between selective effector SMADs CHMI 4237 E - Winter 2010 12 R-SMADS R-SMADS are specific to particular TGF family receptors TbR-I (L45 loop) binds the L3 loop of the MH2 domain of R-SMADS; This ensures specificity of interaction The phosphorylated GS sequence also binds the basic pocket of the R-SMAD (this is the on-off signal); Upon R-SMAD phosphorylation, the SxS sequence binds the basic pocket, weakening the interaction of R-SMADs with their cytoplasmic anchors and Signal Transduction. 2nd edition. favoring oligomerization of 2 R-SMADs with SMAD 4; 2009. Academic Press CHMI 4237 E - Winter 2010 http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf 13 Signal Transduction. 2nd edition. 2009. Academic Press R-SMADS/SMAD 4 Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010 14 Nuclear export and import Ran Ran GDP GDP Ran Ran GDP GDP Ran-GAP Ran GDP Ran GTP Ran GDP Ran GTP RCC1 (Ran GEF) Ran Ran GTP GTP Ran Ran GTP GTP NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1 CHMI 4237 E - Winter 2010 15 SMAD 4 and nuclear export SMAD4 doesn’t have a SxS sequence and thus is not phosphorylated by TbR-I; It also has a nuclear export sequence, which keeps it in the cytosol: ◦ CRM1 binds the NES and mediates interaction with nucleoporins; NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1 Heteromerization with RSMADs masks the NES, allowing SMAD4 to accumulate in the nucleus. CHMI 4237 E - Winter 2010 16 SMAD 4 and nuclear export THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21329–21336, 2005 CHMI 4237 E - Winter 2010 17 Gene modulation by SMADs Signal Transduction. 2nd edition. 2009. Academic Press R-SMAD/SMA4 4 blunts the expression of c-myc through binding a « TGFb inhibitory element » (TIB) in the c-myc promoter; This releases the inhibition on p21CIP expression; R-SMAD/SMAD 4 also interacts with several transcription factors to promote CKI gene transcirption, leading to cell cycle inhition. CHMI 4237 E - Winter 2010 18 Gene modulation by SMADs Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010 19 Modulation of SMAD Activity Dephosphorylation of SMADs in the nucleus leads to their export to the cytosol; Phosphorylation of the linker region of SMADs promote their regulation; Phosphorylation by CDKs and MAPKs lead to cytosolic retention and degradation of SMADs Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010 20 SMURFs Oncogene (2004) 23, 2071–2078 C2 domains phospholipid-binding WW domains mediate protein-protein interaction HECT domain: E3 ubiquitin ligase activity CHMI 4237 E - Winter 2010 21 SMURFs Oncogene (2004) 23, 6914–6923 Oncogene (2004) 23, 2071–2078 CHMI 4237 E - Winter 2010 22 Modulation by inhibitory SMADs In the absence of TGFb, both are retained in the nucleus; SMAD6 and SMAD7 are up-regulated and exported into the cytosol following TGFb signalling; SMAD 6 competes with SMAD4 for R-SMAD1 binding; SMAD7 binds with SMURF2 and mediates the degradation of TbR-I; Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010 23