1. CHMI 4237 E Special topics in Biochemistry Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry Laurentian University Cell proliferation 4- Signaling to the cell cycle – TGF- 1CHMI 4237 E - Winter 2010

2. 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 20102

3. 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; CHMI 4237 E - Winter 20103 M.B. Sporn / Cytokine & Growth Factor Reviews 17 (2006) 3–7 + EGF+ PDGF SGF

4. Transforming growth factor beta Family of over 33 proteins, which includes: ◦TGF ◦Bone morphogenetic proteins (BMPs) ◦Activins ◦Growth and differentiation factors (GDFs) Number of effects: ◦Proliferation (stimulation/inhibtion) ◦Differentiation ◦Cell adhesion ◦Cell migration ◦Cell death CHMI 4237 E - Winter 20104 nature cell biology volume 9 | number 9 | SEPTEMBER 2007

5. TGF- secretion TGF- is first synthesized on the ribosome as a pre-pro- protein; The pre-sequence is removed during insertion into the ER lumen During its transit in the secretory pathway, TGF- 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) CHMI 4237 E - Winter 20105 http://www.comparative-hepatology.com/content/figures/1476-5926-6-7-6.jpg

6. TGF- receptor CHMI 4237 E - Winter 20106 TGF- 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 TR-I subunit : ◦possess a 30-amino acid GS domain preceding the kinase domain TR-II subunit: Activates receptor in a ligand-specific manner by phosphorylating the GS sequence of TR-I Doesn’t have a GS sequence; T  R-IT  R-II http://jkweb.berkeley.edu/external/pdb/2001/tgf _beta_R1/receptor_schematic.jpg

7. TR-I activation CHMI 4237 E - Winter 20107 In the absence of ligand: TR-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; TR-I is stabilized in this form through the binding of FKBP12; http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg

8. TR-I activation CHMI 4237 E - Winter 20108 TGF binding causes the dimerization of TR-I and TR-II; TR-II phosphorylates the GS sequence; This is sufficient to dislodge the GS sequence from the N- lobe and allow ATP binding; Signal Transduction. 2 nd edition. 2009. Academic Press

9. TR-I activation CHMI 4237 E - Winter 20109 Phosphorylated TR-I acts as a docking site for the actual signal transducers: a family of proteins called R-SMADS; SMADS are brought to the TR-I/TR-II dimer by a membrane- bound protein called SARA; R-SMAD phosphorylation by TR-I triggers the signaling cascade. http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg

10. SMADS CHMI 4237 E - Winter 201010 Three classes are recognized: ◦R-SMAD: initiate signaling at the TR; ◦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 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 TR-I Signal Transduction. 2 nd edition. 2009. Academic Press

11. SMADS CHMI 4237 E - Winter 201011 http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf

12. SMADS CHMI 4237 E - Winter 201012 Signal Transduction. 2 nd edition. 2009. Academic Press When phosphorylated by TR-I, the SxS motif interacts with a basic pocket in MH2; This promotes heteromerization between selective effector SMADs

13. R-SMADS CHMI 4237 E - Winter 201013 R-SMADS are specific to particular TGF family receptors TR-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 favoring oligomerization of 2 R-SMADs with SMAD 4; http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf Signal Transduction. 2 nd edition. 2009. Academic Press

14. R-SMADS/SMAD 4 CHMI 4237 E - Winter 201014 Signal Transduction. 2 nd edition. 2009. Academic Press

15. Nuclear export and import CHMI 4237 E - Winter 201015 Ran-GAP RCC1 (Ran GEF) Ran GDP Ran GTP Ran GDP Ran GTP Ran GDP NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1

16. SMAD 4 and nuclear export CHMI 4237 E - Winter 201016 SMAD4 doesn’t have a SxS sequence and thus is not phosphorylated by TR-I; It also has a nuclear export sequence, which keeps it in the cytosol: ◦CRM1 binds the NES and mediates interaction with nucleoporins; Heteromerization with R- SMADs masks the NES, allowing SMAD4 to accumulate in the nucleus. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1

17. SMAD 4 and nuclear export CHMI 4237 E - Winter 201017 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21329–21336, 2005

18. Gene modulation by SMADs CHMI 4237 E - Winter 201018 R-SMAD/SMA4 4 blunts the expression of c-myc through binding a « TGF inhibitory element » (TIB) in the c-myc promoter; This releases the inhibition on p21 CIP expression; R-SMAD/SMAD 4 also interacts with several transcription factors to promote CKI gene transcirption, leading to cell cycle inhition. Signal Transduction. 2 nd edition. 2009. Academic Press

19. Gene modulation by SMADs CHMI 4237 E - Winter 201019 Signal Transduction. 2 nd edition. 2009. Academic Press

20. Modulation of SMAD Activity CHMI 4237 E - Winter 201020 Signal Transduction. 2 nd edition. 2009. Academic Press 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

21. SMURFs CHMI 4237 E - Winter 201021 Oncogene (2004) 23, 2071–2078 C2 domains  phospholipid-binding WW domains  mediate protein-protein interaction HECT domain: E3 ubiquitin ligase activity

22. SMURFs CHMI 4237 E - Winter 201022 Oncogene (2004) 23, 2071–2078 Oncogene (2004) 23, 6914–6923

23. Modulation by inhibitory SMADs CHMI 4237 E - Winter 201023 Signal Transduction. 2 nd edition. 2009. Academic Press In the absence of TGF, both are retained in the nucleus; SMAD6 and SMAD7 are up-regulated and exported into the cytosol following TGFsignalling; SMAD 6 competes with SMAD4 for R-SMAD1 binding; SMAD7 binds with SMURF2 and mediates the degradation of TR-I;