CHMI 4237 E Special topics in Biochemistry

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CHMI 4237 ESpecial topics in Biochemistry
Cell proliferation 4- Signaling to the cell cycle
TGF-b

• Eric R. Gauthier, Ph.D.
• Dept. Chemistry-Biochemistry
• Laurentian University

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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?

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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

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Transforming growth factor beta

• 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

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TGF-b secretion

• 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)

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TGF-b receptor

• 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
• 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
• Doesnt have a GS sequence

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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

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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
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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
  membrane-bound protein called SARA
• R-SMAD phosphorylation by TbR-I triggers the
  signaling cascade.

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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
• 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

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SMADS
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SMADS

• When phosphorylated by TbR-I, the SxS motif
  interacts with a basic pocket in MH2
• This promotes heteromerization between selective
  effector SMADs

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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
  favoring oligomerization of 2 R-SMADs with SMAD
  4

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R-SMADS/SMAD 4
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Nuclear export and import
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SMAD 4 and nuclear export

• SMAD4 doesnt 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.

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SMAD 4 and nuclear export
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Gene modulation by SMADs

• 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.

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Gene modulation by SMADs
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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

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SMURFs

• C2 domains ? phospholipid-binding
• WW domains? mediate protein-protein interaction
• HECT domain E3 ubiquitin ligase activity

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SMURFs
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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