CHMI 4237 E Special topics in Biochemistry

CHMI 4237 ECHMI 4237 E

Special topics in Special topics in BiochemistryBiochemistry

Eric R. Gauthier, Ph.D.Dept. Chemistry-Biochemistry

Laurentian University

Cell proliferation4- Signaling to the cell cycle – TGF-

1CHMI 4237 E - Winter 2010

So, what are the BIG questions: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 betaTransforming 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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M.B. Sporn / Cytokine & Growth Factor Reviews 17 (2006) 3–7

+ EGF + PDGF

SGF

Transforming growth factor betaTransforming 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

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nature cell biology volume 9 | number 9 | SEPTEMBER 2007

TGF-TGF- secretion 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)

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http://www.comparative-hepatology.com/content/figures/1476-5926-6-7-6.jpg

TGF-TGF- receptor receptor

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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

TR-I subunit: ◦ possess a 30-amino acid GS domain

preceding the kinase domain

TR-II subunit: Activates receptor in a ligand-specific

manner by phosphorylating the GS sequence of TR-I

Doesn’t have a GS sequence;

TR-ITR-II

http://jkweb.berkeley.edu/external/pdb/2001/tgf_beta_R1/receptor_schematic.jpg

TTR-I activationR-I activation

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In the absence of ligand: TR-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;

TR-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

TTR-I activationR-I activation

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TGF binding causes the dimerization of TR-I and TR-II;

TR-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

TTR-I activationR-I activation

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Phosphorylated TR-I acts as a docking site for the actual signal transducers: a family of proteins called R-SMADS;

SMADS are brought to the TR-I/TR-II dimer by a membrane-bound protein called SARA;

R-SMAD phosphorylation by TR-I triggers the signaling cascade.

http://www.cellbiol.net/layout/imagesBook/groot/20.05%20schematic%20view%20receptor%20activation.jpg

SMADSSMADS

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Three classes are recognized:◦ R-SMAD: initiate signaling at the TR;◦ 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 TR-I

Signal Transduction. 2nd edition. 2009. Academic Press

SMADSSMADS

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http://www.nature.com/nature/journal/v425/n6958/pdf/nature02006.pdf

SMADSSMADS

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Signal Transduction. 2nd edition. 2009. Academic Press

When phosphorylated by TR-I, the SxS motif interacts with a basic pocket in MH2;

This promotes heteromerization between selective effector SMADs

R-SMADSR-SMADS

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R-SMADS are specific to particular TGF family receptors

TR-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://ww

w.nature.com

/nature/journal/v425/n6958/pdf/nature02006.pdfSignal Transduction. 2nd edition. 2009. Academic Press

R-SMADS/SMAD 4R-SMADS/SMAD 4

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Signal Transduction. 2nd edition. 2009. Academic Press

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Nuclear export and importNuclear export and import

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Ran-GAP

RCC1(Ran GEF)

Ran GDP

Ran GTP

Ran GDPRan

GDP

Ran GDP

Ran GDP

Ran GTPRan

GTPRan GTP

Ran GTP

Ran GTP

Ran GDP

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | MARCH 2004 | 1

SMAD 4 and nuclear exportSMAD 4 and nuclear export

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SMAD4 doesn’t have a SxS sequence and thus is not phosphorylated by TR-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

SMAD 4 and nuclear exportSMAD 4 and nuclear export

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 22, Issue of June 3, pp. 21329–21336, 2005

Gene modulation by SMADsGene modulation by SMADs

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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 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 SMADsGene modulation by SMADs

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

Modulation of SMAD ActivityModulation of SMAD Activity

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Signal Transduction. 2nd 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

SMURFsSMURFs

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Oncogene (2004) 23, 2071–2078

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

SMURFsSMURFs

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Oncogene (2004) 23, 2071–2078

Oncogene (2004) 23, 6914–6923

Modulation by inhibitory SMADsModulation by inhibitory SMADs

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Signal Transduction. 2nd 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 TGFsignalling;

SMAD 6 competes with SMAD4 for R-SMAD1 binding;

SMAD7 binds with SMURF2 and mediates the degradation of TR-I;