Transcript 5 - cellbiochem.ca

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