A brief note on two pathways related to cell signalling.

1. CELL SIGNALLING PATHWAYS
AND
NETWORKS

2. TGF-β PATHWAY
&
INOSITOL PHOSPHATE PATHWAY
By:
GIRISH KUMAR K
IV MSc
BIOMEDICAL SCIENCE

3. TGF – β PATHWAY
• The Transforming growth factor beta (TGF – β) signaling
pathway is involved in many cellular processes in both the adult
organism and the developing embryo including cell growth, cell
differentiation, apoptosis, cellular homeostasis and other cellular
functions.
• In spite of the wide range of cellular processes that the TGF – β
signaling pathway regulates, the process is relatively simple.
• TGF – β superfamily ligands bind to a type II receptor, which
recruits and phosphorylates a type I receptor.
• The type I receptor then phosphorylates receptor-regulated
SMADs (R-SMADs), a intracellular proteins which can now bind
the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate
in the nucleus where they act as transcription factors and
participate in the regulation of target gene expression.

4. MECHANISM
 LIGAND BINDING
 RECEPTOR RECRUITMENT AND
PHOSPHORYLATION
 SMAD PHSOPHORYLATION
 CoSMAD PHOSPHORYLATION
 TRANSCRIPTION

5. LIGAND BINDING
The TGF – β superfamily of ligands includes:
 Bone morphogenetic proteins (BMPs)
 Growth and differentiation factors (GDFs)
 Anti-müllerian hormone (AMH)
 Activin
 Nodal
 TGF – β's.

6. • Signalling begins with the binding of a TGF – β
superfamily ligand to a TGF – β type II receptor.
• The type II receptor is a serine/threonine
receptor kinase, which catalyses the
phosphorylation of the Type I receptor.
• Each class of ligand binds to a specific type II
receptor.
• In mammals there are seven known type I
receptors and five type II receptors.

7. • There are three activins – Activin A, Activin B
and Activin AB. Activins.
• They are involved in embryogenesis and
osteogenesis. They also regulate many hormones
including pituitary, gonadal and hypothalamic
hormones as well as insulin. They are also nerve
cell survival factors.

8. • The BMPs bind to the Bone morphogenetic protein
receptor type-2 (BMPR2).
• They are involved in a multitude of cellular functions
including osteogenesis, cell differentiation,
anterior/posterior axis specification, growth, and
homeostasis.
• The TGF beta family include: TGFβ1, TGFβ2, TGFβ3.
• Like the BMPS, TGF βs are involved in embryogenesis
and cell differentiation, but they are also involved in
apoptosis, as well as other functions.
• They bind to TGF-β receptor type-2 (TGFBR2).

9. LIGAND BINDING

10. • The TGF β ligand binds to a type II receptor dimer,
which recruits a type I receptor dimer forming a
hetero-tetrameric complex with the ligand.
• These receptors are serine/threonine kinase receptors.
• They have a cysteine rich extracellular domain, a
transmembrane domain and a cytoplasmic
serine/threonine rich domain.
• The GS domain of the type I receptor consists of a series
of about thirty serine-glycine repeats. The binding of a
TGF β family ligand causes the rotation of the receptors
so that their cytoplasmic kinase domains are arranged
in a catalytically favorable orientation.
• The Type II receptor phosphorylates serine residues of
the Type I receptor, which activates the protein.
RECEPTOR RECRUITMENT AND
PHOSPHORYLATION

11. RECEPTOR RECRUITMENT AND
PHOSPHORYLATION

12. SMAD PHOSPHORYLATION
• There are five receptor regulated SMADs:
SMAD1, SMAD2, SMAD3, SMAD5, and SMAD9
(sometimes referred to as SMAD8).
• There are essentially two intracellular pathways
involving these R-SMADs.
1. TGF beta's, Activins, Nodals and some GDFs are
mediated by SMAD2 and SMAD3.
2. BMPs, AMH and a few GDFs are mediated by
SMAD1, SMAD5 and SMAD9.

13. • The binding of the R-SMAD to the type I
receptor is mediated by a zinc double finger
FYVE domain containing protein. Two such
proteins that mediate the TGF beta pathway
include SARA (The SMAD anchor for receptor
activation) and HGS (Hepatocyte growth factor-
regulated tyrosine kinase substrate).

14. • SARA is present in an early endosome which, by
clathrin-mediated endocytosis, internalizes the
receptor complex.
• SARA recruits an R-SMAD. SARA permits the
binding of the R-SMAD to the L45 region of the
Type I receptor.
• SARA orients the R-SMAD such that serine residue
on its C-terminus faces the catalytic region of the
Type I receptor.
• The Type I receptor phosphorylates the serine
residue of the R-SMAD.
• Phosphorylation induces a conformational change
in the MH2 domain of the R-SMAD and its
subsequent dissociation from the receptor complex
and SARA.

15. SMAD PHOSPHORYLATION

16. CoSMAD BINDING
• The phosphorylated RSMAD has a high
affinity for a coSMAD (e.g. SMAD4) and
forms a complex with one.
• The phosphate group does not act as a
docking site for coSMAD, rather the
phosphorylation opens up an amino acid
stretch allowing interaction.

17. CoSMAD BINDING

18. TRANSCRIPTION
• The phosphorylated RSMAD/coSMAD complex enters the
nucleus where it binds transcription promoters/cofactors and
causes the transcription of DNA.
• Bone morphogenetic proteins cause the transcription of
mRNAs involved in osteogenesis, neurogenesis, and ventral
mesoderm specification.
• TGF βs cause the transcription of mRNAs involved in
apoptosis, extracellular matrix neogenesis and
immunosuppression. It is also involved in G1 arrest in the cell
cycle.
• Activin causes the transcription of mRNAs involved in
gonadal growth, embryo differentiation and placenta
formation.
• Nodal causes the transcription of mRNAs involved in left and
right axis specification, and mesoderm and endoderm
induction.

19. TRANSCRIPTION

20. PATHWAY REGULATION
• The TGF β signaling pathway is involved in a
wide range of cellular process and subsequently
is very heavily regulated.
• There are a variety of mechanisms that the
pathway is modulated both positively and
negatively:
1. There are agonists for ligands and R-SMADs.
2. There are decoy receptors .
3. R-SMADs and receptors are ubiquitinated.

21. LIGAND AGONISTS/ANTAGONISTS
• Both chordin and noggin are antagonists of BMP's. They bind
BMP's preventing the binding of the ligand to the receptor.
• It has been demonstrated that Chordin and Noggin dorsalize
mesoderm.
• They are both found in the dorsal lip of Xenopus and convert
otherwise epidermis specified tissue into neural tissue.
• Noggin plays a key role in cartilage and bone patterning. Mice
Noggin have excess cartilage and lacked joint formation.
• Members of the DAN family of proteins also antagonize TGF
beta family members. They include Cerberus, DAN, and
Gremlin. These proteins contain nine conserved cysteines
which can form disulfide bridges. It is believed that DAN
antagonizes GDF5, GDF6 and GDF7.

22. • Follistatin inhibits Activin, which it binds. It directly
affects follicle-stimulating hormone (FSH) secretion.
Follistatin also is implicated in prostate cancers
where mutations in its gene may preventing it from
acting on activin which has anti-proliferative
properties.
• Lefty is a regulator of TGFβ and is involved in the
axis patterning during embryogenesis. It is also a
member of the TGF superfamily of proteins. It is
asymmetrically expressed in the left side of murine
embryos and subsequently plays a role in left-right
specification. Lefty acts by preventing the
phosphorylation of R-SMADs.
• Drug-based antagonists have also been identified,
such as SB431542, which selectively inhibits ALK4,
ALK5, and ALK7.

23. INOSITOL PHOSPHATE PATHWAY

24. INOSITOL TRISPHOSPHATE
• Inositol trisphosphate (or inositol 1,4,5-
trisphosphate) together
with diacylglycerol(DAG) is a secondary
messenger molecule used in signal
transduction and lipid signaling in biological
cells.
• While DAG stays inside the membrane, IP3 is
soluble and diffuses through the cell. It is made
by hydrolysis of phosphatidylinositol 4,5-
bisphosphate (PIP2), a phospholipid that is
located in the plasma membrane,
by phospholipase C (PLC).

25. INOSITOL PHOSPHATE METABOLISM
• The Ca2+ -mobilizing messenger function of inositol
1,4,5-trisphosphate (InsP3) is terminated through
its metabolism by a complex inositol phosphate
metabolic pathway that has two main functions.
• Firstly, it produces free inositol that is resynthesized
to PtdIns (Phosphatidylinositol), which can be
reused for further signalling.
• Secondly, it generates an assortment of inositol
phosphates, some of which contribute to the
multipurpose inositol polyphosphate signalling
pathway.

26. • The Ins1,4,5P3 that enters the metabolic
pathway is metabolized via two pathways. It is
dephosphorylated by Type I inositol
polyphosphate 5-phosphatase to form Ins1,4P2
or it can be phosphorylated by InsP3 3- kinase
to form Ins1,3,4,5P4.
• Metabolism of the inositol phosphates is carried
out by a large number of inositol phosphate
kinases and phosphatases.

27. • The process of inositol phosphate metabolism generates a
large number of inositol phosphates. Many of these are
metabolic intermediates, but some have been implicated
in a variety of control functions:
• Ins1,4P2
There is some evidence to suggest that Ins1,4P2 may
function within the nucleus to activate DNA polymerase.
This inositol phosphate has also been implicated in Ca2+
signalling and cardiac hypertrophy.
• Ins1,3,4P3
This inositol phosphate has an important signalling
function as a negative regulator of the Ins1,3,4,5,6P5 1-
phosphatase that controls the level of Ins3,4,5,6P4, which
is an inhibitor of Ca2+ -sensitive Cl- channels.
INOSITOL PHOSPHATE PATHWAY

28. Ins3,4,5,6P4
Ins3,4,5,6P4 functions as an inhibitor of the
Ca2+ -sensitive Cl − channels (CLCAs) in
epithelial cells, which regulate salt and fluid
secretion, cell volume homoeostasis and
electrical excitability in neurons and smooth
muscle cells. It appears to act by preventing Ca2+
/calmodulin-dependent protein kinase II
(CaMKII) from activating the channel.
Ins3,4,5,6P4 is formed by an Ins1,3,4,5,6P5 1-
phosphatase, which is activated by Ins1,3,4P3.

29. InsP6
There are a number of suggestions concerning the
possible messenger role of InsP6. Cells contain high
levels of InsP6 (approximately 15-100 μM), and
much of this is probably not in solution, but is
probably attached to the phospholipids in
membranes through electrostatic interactions with
bivalent cations. The level of InsP6 does not change
much during acute stimulation, but its level can
vary over the long term, as occurs during the cell
cycle and during cellular differentiation.

30. Trafficking of vesicles
InsP6 may inhibit clathrin cage assembly by
binding to adaptor protein (AP)-2 and -3. Such a
role in membrane trafficking is also consistent
with the observation that InsP6 binds to
synaptotagmin by competing with the inositol
lipid-binding site. It therefore seems that InsP6
may function as a negative regulator of
endocytic vesicle traffic. Such a possibility may
explain the observation that GRAB, which is a
guanine nucleotide exchange factor that acts on
Rab3A, interacts with InsP6 kinase which
converts InsP6 into InsP7.

31. Endocytosis
In insulin-secreting β-cells, InsP6 appears to
promote dynamin I-mediated endocytosis
through a mechanism that depends upon
protein kinase C (PKC), which may act to inhibit
the phosphoinositide phosphatase synaptojanin,
thereby raising the level of PtdIns4,5P2 that has
been implicated in vesicle dynamics.

32. Regulation of Ca2+ channels
InsP6 is present in many brain regions and
appears to be elevated following neural activity.
It may act by stimulating adenylyl cyclase to
produce cyclic AMP, which then increases the
activity of L-type Ca2+ channels. Alternatively, it
may activate L-type Ca2+ channels through an
inhibition of protein phosphatases. In vascular
smooth muscle cells, InsP6 appears to act
through a protein kinase C (PKC)-dependent
pathway.

33. Regulation of gene transcription
Studies on yeast have revealed that ARG82,
which is an Ins1,4,5P3 6-kinase that
phosphorylates Ins1,4,5P3 within the nucleus to
form Ins1,4,5,6P4, functions as a transcriptional
regulator.

34. Regulation of mRNA export from the nucleus
Studies on yeast indicate that InsP6 formed by
an inositol polyphosphate kinase located on
the nuclear pores may facilitate the export of
mRNA from the nucleus.

35. THANK YOU