CHMI 4237 E Special topics in Biochemistry

1. CHMI 4237 ESpecialtopics in Biochemistry Cellproliferation 4- Signaling to the cell cycle – TGF-b Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry LaurentianUniversity CHMI 4237 E - Winter 2010

2. 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? So, what are the BIG questions: CHMI 4237 E - Winter 2010

3. Isolated as a component of « sarcoma growth factor »; • Triggers a number of biological effects, including cell proliferation and cell cycle arrest; SGF + EGF + PDGF Transforminggrowth factor beta CHMI 4237 E - Winter 2010 M.B. Sporn / Cytokine & Growth Factor Reviews 17 (2006) 3–7

4. Family of over 33 proteins, which includes: • TGFb • Bone morphogenetic proteins (BMPs) • Activins • Growth and differentiation factors (GDFs) • Number of effects: • Proliferation (stimulation/inhibtion) • Differentiation • Cell adhesion • Cell migration • Cell death nature cell biology volume 9 | number 9 | SEPTEMBER 2007 Transforminggrowth factor beta CHMI 4237 E - Winter 2010

5. TGF-b 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-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) TGF-bsecretion http://www.comparative-hepatology.com/content/figures/1476-5926-6-7-6.jpg CHMI 4237 E - Winter 2010

6. TGF-b triggers itseffects on the cell by causing the dimerization of twosubunits of the TGF receptor: • Single-span membrane proteins • PossessSer/Thr kinaseactivity • TbR-I subunit: • possess a 30-aminoacid GS domainpreceding the kinase domain • TbR-II subunit: • Activatesreceptor in a ligand-specificmanner by phosphorylating the GS sequence of TbR-I • Doesn’t have a GS sequence; TbR-II TbR-I TGF-breceptor http://jkweb.berkeley.edu/external/pdb/2001/tgf_beta_R1/receptor_schematic.jpg CHMI 4237 E - Winter 2010

7. 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; TbR-I activation CHMI 4237 E - Winter 2010 http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg

8. 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; TbR-I activation Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010

9. 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 membrane-bound protein called SARA; • R-SMAD phosphorylation by TbR-I triggers the signaling cascade. TbR-I activation CHMI 4237 E - Winter 2010 http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg

10. 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 • 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 SMADS Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010

11. http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdfhttp://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf SMADS CHMI 4237 E - Winter 2010

12. When phosphorylated by TbR-I, the SxS motif interacts with a basic pocket in MH2; • This promotes heteromerization between selective effector SMADs Signal Transduction. 2nd edition. 2009. Academic Press SMADS CHMI 4237 E - Winter 2010

13. 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 favoring oligomerization of 2 R-SMADs with SMAD 4; http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf R-SMADS Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010

14. Signal Transduction. 2nd edition. 2009. Academic Press Signal Transduction. 2nd edition. 2009. Academic Press R-SMADS/SMAD 4 CHMI 4237 E - Winter 2010

15. Ran GDP Ran GDP Ran GDP Ran GDP Ran-GAP Ran GDP Ran GTP Ran GTP Ran GDP RCC1 (Ran GEF) Nuclear export and import Ran GTP Ran GTP Ran GTP Ran GTP CHMI 4237 E - Winter 2010 NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1

16. 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; • Heteromerization with R-SMADs masks the NES, allowing SMAD4 to accumulate in the nucleus. SMAD 4 and nuclear export NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1 CHMI 4237 E - Winter 2010

17. 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

18. 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. Signal Transduction. 2nd edition. 2009. Academic Press Gene modulation by SMADs CHMI 4237 E - Winter 2010

19. Gene modulation by SMADs CHMI 4237 E - Winter 2010 Signal Transduction. 2nd edition. 2009. Academic Press

20. 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 Modulation of SMAD Activity Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010

21. C2 domains  phospholipid-binding • WW domains mediate protein-protein interaction • HECT domain: E3 ubiquitin ligase activity Oncogene (2004) 23, 2071–2078 SMURFs CHMI 4237 E - Winter 2010

22. Oncogene (2004) 23, 6914–6923 SMURFs Oncogene (2004) 23, 2071–2078 CHMI 4237 E - Winter 2010

23. 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; Modulation by inhibitorySMADs Signal Transduction. 2nd edition. 2009. Academic Press CHMI 4237 E - Winter 2010