Biologia Molecular e Celular

2º Semestre
Ano Lectivo 2011-2012

Signaling pathways
  There are number of different types of signaling pathways that
lead to the activation of various target proteins from transcription
factors that ultimately lead to changes in gene expression to
growth factors that directly mediate changes in biological process
such as growth.
  We have already talked about pathways that alter the intracellular
concentration of calcium and other 2nd messenger mediated
pathways.
  Now we are going to briefly discuss 2 additional pathways that
mediate numerous biological processes through the activation of
downstream target genes.

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MAP Kinase Pathways
  The central components of the this pathway are serine/threonine
kinases called MAP Kinases (or mitogen activated protein kinases).
  They are typically activated in response to growth factors and
other signaling molecules and function to regulate several cellular
response such as cell growth and differentiation.
  In this pathway, stimulation of growth factor receptors leads to the
activation of the small GTP binding protein Ras.

MAP Kinase Pathways

  Ras proteins belong to a large family of about 50 related proteins
that are called small GTP binding proteins. However they behave
in a similar manner to the G proteins.
  Ras, in turn, interacts with the Raf protein kinase. Activated Raf
then phosphorylates and activates MEK (MAP/ERK kinase).
  MEK is a dual specificity protein kinase that activates ERK by
phosphorylation on threonine and tyrosine residues that are
separated by one amino acid.
  ERK in turn phosphorylates a variety of nuclear and cytoplasmic
target proteins such as other proteins kinases and transcription
factors.

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MAP Kinase Pathways
Stimulation of growth factor receptors leads to
activation of the small GTP-binding protein Ras,
which interacts with the Raf protein kinase.

Raf phosphorylates and activates MEK, a dual-

specificity protein kinase that activates ERK
by phosphorylation on both threonine and
tyrosine residues (Thr-183 and Tyr-185).

ERK then phosphorylates a variety of

nuclear and cytoplasmic target proteins.
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MAP Kinase Pathways

  Most of what we know today about this pathway in mammalian
cells has emerged from studies done on the Ras proteins.
  Ras was the first identified oncogenic protein of tumor viruses.
  It was identified as the cause of the Rat Sarcoma virus.
  Today, mutations in the Ras gene are found in numerous different
types of cancers.
  In addition to its role in cancer, numerous experts have led to the
knowledge that the activated Ras protein is crucial for growth
factor-induced proliferation and survival of normal cells too.
  Ras proteins are guanine nucleotide binding proteins that function
similar to that of the alpha subunit of G proteins. However, Ras
functions as a monomer, not in association with other proteins like
G proteins.
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MAP Kinase Pathways
  Activation of Ras is mediated by guanine nucleotide exchange
factors (GEF) that stimulate the release of bound GDP and its
exchange for GTP.
  Activity of Ras-GTP complex is then terminate by hydrolysis of GTP
which causes the interaction of Ras-GTP with GTPase activating
proteins.
  What happens is the role of Ras in Cancer?
  Why do so many cancers express mutated version of the
Ras protein?
  The mutations of Ras genes in human cancers have the effect of
inhibiting GTP hydrolysis by the Ras proteins.
  These mutated Ras proteins therefore remain continuously in the active
GTP bound form which drives the unregulated proliferation of cancer
cells in the absence of growth factor stimulation.
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MAP Kinase Pathways

  The Ras proteins are guanine nucleotide-binding proteins that
function analogously to the α subunits of G proteins, alternating
between inactive GDP-bound and active GTP-bound forms.
  In contrast to the G protein α subunits, however, Ras functions as
a monomer rather than in association with βγ subunits.
  Ras activation is mediated by guanine nucleotide exchange factors
that stimulate the release of bound GDP and its exchange for GTP.
  Activity of the Ras-GTP complex is then terminated by GTP
hydrolysis, which is stimulated by the interaction of Ras-GTP with
GTPase-activating proteins.

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Regulation of
Ras proteins

Ras proteins
alternate
between inactive
GDP-bound and
active GTP-
bound states.

The Cell: A Molecular

Approach. 2nd edition.
Cooper GM. Sinauer
Associates. Figure 13.33.

MAP Kinase Pathways

  It is interesting to note that the mutations of ras genes in human
cancers have the effect of inhibiting GTP hydrolysis by the Ras
proteins.
  These mutated Ras proteins therefore remain continuously in the
active GTP-bound form, driving the unregulated proliferation of
cancer cells even in the absence of growth factor stimulation.

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 MAP Kinase Pathways
  The best understood mode of Ras activation is that mediated by
receptor protein-tyrosine kinases.
  Autophosphorylation of these receptors results in their association
with Ras guanine nucleotide exchange factors as a result of SH2-
mediated protein interactions.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.34. 11

Ras activation downstream of receptor protein-tyrosine kinases

A complex of Grb2 and the guanine nucleotide exchange factor Sos binds to
a phosphotyrosine-containing sequence in the activated receptor via the Grb2
SH2 domain.
This interaction recruits Sos to the plasma membrane, where it can stimulate
Ras GDP/GTP exchange. The activated Ras-GTP complex then binds to
the Raf protein kinase.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.34.

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 MAP Kinase Pathways
  The guanine nucleotide exchange factor Sos, which is bound to the
SH2-containing protein Grb2 in the cytosol of unstimulated cells.
  Tyrosine phosphorylation of receptors (or of other receptor-
associated proteins) creates a binding site for the Grb2 SH2
domains.
  Association of Grb2 with activated receptors localizes Sos to the
plasma membrane, where it is able to interact with Ras proteins.

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MAP Kinase Pathways

  Ras proteins are anchored to the inner leaflet of the
plasma membrane by lipids attached to the Ras C
terminus.
  Sos then stimulates guanine nucleotide exchange,
resulting in formation of the active Ras-GTP complex.
  In its active GTP-bound form, Ras interacts with a
number of effector proteins, including the Raf protein-
serine/threonine kinase.
  This interaction with Ras recruits Raf from the cytosol
to the plasma membrane, where it is activated as a
result of phosphorylation by both protein-tyrosine and
protein-serine/threonine kinases.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 12.10.
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 MAP Kinase Pathways
  The best understood mode of Ras activation
occurs through receptor tyrosine kinase as is
seen in the orange pathway.
  However, most eukaryotic cells including
mammalian cells have multiple different MAP
kinase pathways that control very distinct
cellular responses.
  Regardless of the biological response each
pathway generally consists of 3 protein
kinases: a terminal MAP kinase and 2
upstream kinases the regulate activity of the
terminal kinase. These upstream kinases
would be similar to the Raf and Mek.

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MAP Kinase Pathways

  In mammalian cells 3 different MAP
kinase have been identified: These are
ERK, p38 and JNK kinases.
  The latter 2 kinases are preferentially
activated in response to
inflammatory cytokines and cellular
stress.
  As such they are activated by the Rho
family of small GTP binding proteins
instead of Ras.
  Ultimately, these amount of diversity in
MAP kinase proteins and pathways
allows eukaryotic cells to regulate
cellular responses to numerous
environment signals or challenges.

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NF-kB Signaling
  This is a pathway that specifically targets a certain family of
transcription factors.
  The NF-kB family consists of 5 transcription factors that have
key roles:
  in the immune system (mediate the responses of various
cytokines, including interleukin 1 (IL-1) and the tumor necrosis
factor α (TNF-α)
  in inflammation as well
  in regulation of proliferation and survival of many types of animal
cells.

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NF-kB Signaling
  In mammals there are 5 proteins NF-kB: RelA, RelB, c-Rel,
p50, p52
  They form homo-and heterodimers and each activates a
specific group of genes
  Inhibitory proteins, IkB, bind dimers to keep them inactive in
the cytoplasm
  Members of TF family are activated in response to various
stimuli including cytokines, GFs, viral infection and DNA
damage.

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 NF-kB Signaling
  Activation of NF-kB
results from signals that
activate the IkB kinase
which phosphorylates IkB.
  The protein
phosphorylated IkB
undergoes ubiquitination
and degradation by
proteasome.
  The dimers NF-kB
migrates to the nucleus
(the nuclear localization
signal is exposed)
and stimulate the
transcription of specific
genes.

  NfkB is activated by signals that lead to

proteolysis of the inhibitory IkB which allows
NF-kB to translocate to the nucleus to
induce expression of its target genes.
  One of the target genes induced by Nf-KB is
IkB which means that NF-KB signaling leads
to the synthesis of IkB. When enough IkB is
present, it results in inhibiting continued Nf-
KB activity.
  This kind of mechanism is very important
because the extent and duration of NF-kB
activity can determine the transcriptional
response of the cell.
  Some target genes are induced by
transient NF-kB activity while others
require several hours of NF-kB
signaling.

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Life, death and apoptosis
  Apoptosis from the Greek meaning "fall"
  The number of cells in multicellular organisms is
controlled by:
  rate of cell division
  rate of cell death

  In adult tissues cell death balances cell division

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Life, death and apoptosis

  Necrosis: cell death process caused by an acute injury
(a cell swells and bursts releasing their contents on
neighboring cells). It causes a harmful inflammatory
response.
  Apoptosis: the cell dies orderly without interfering
with the neighboring cells.

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 Life, death and apoptosis
  In contrast to the accidental
death of cells that results from an
acute injury, programmed cell
death is an active process
characterized by a distinct
morphological change known as
apoptosis.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.47.
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Apoptosis
  During apoptosis, chromosomal DNA is usually
fragmented as a result of cleavage between nucleosomes.
  The chromatin condenses and the nucleus then breaks up
into small pieces.
  The cell itself shrinks and breaks up into membrane-
enclosed fragments called apoptotic bodies.
  Such apoptotic cells and cell fragments are readily
recognized and phagocytosed by both macrophages and
neighboring cells, so cells that die by apoptosis are
efficiently removed from tissues.
  In contrast, cells that die as a result of acute injury swell
and lyse, releasing their contents into the extracellular
space and causing inflammation.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.47. 24

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 Apoptosis

  The cell shrinks and condenses

  The cytoskeleton collapses
  The nuclear envelope is
disassembled and the nuclear
DNA is broken into fragments
  The cell surface undergoes
changes in order to be
phagocytized by cells
neighboring macrophages or
before release of their contents
occur

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer 25

Associates. Figure 13.47.

Apoptosis
  Studies of programmed cell death during the development of C. elegans have
identified three genes that play key roles in regulating and executing apoptosis.

  Two genes, ced-3 and ced-4, are required for apoptosis to occur; if either
of these genes is inactivated, the normal programmed cell deaths do not take
place.

  A third gene, ced-9, functions as a negative regulator of apoptosis.

  If ced-9 is inactivated by mutation, the cells that would normally survive fail to do so.

  Instead, they also undergo apoptosis, leading to death of the developing animal.

  Conversely, if ced-9 is expressed at an abnormally high level, the normal

programmed cell deaths fail to occur

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 Apoptosis
  Genes related to ced-3, ced-4, and ced-9 have been identified in mammals and
found to encode proteins that represent conserved effectors and regulators of
apoptosis induced by a variety of stimuli.
  Many cell death signals induce apoptosis via a conserved pathway of regulators,
adaptors, and caspases.

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The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.48.

Apoptosis
  In C. elegans, the negative regulator Ced-9 inhibits apoptosis by
binding to the adaptor Ced-4. In the absence of inhibition by Ced-9,
Ced-4 binds two molecules of the caspase Ced-3, resulting in
autocleavage and caspase activation.

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The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.48.

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 Apoptosis
  In mammals, regulators of the Bcl-2 family (Ced-9 homologs) act at
the mitochondria to control release of cytochrome c, which is required
for the binding of caspase-9 to the adaptor Apaf-1 (the Ced-4
homolog). Release of cytochrome c from mitochondria thus signals the
activation of caspase-9, which then activates downstream caspases to
induce apoptosis.

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The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.48.

Apoptosis
  It is mediated by proteases called caspases (cysteine present at
a site active and cleave their target proteins in a specific aspartic
acid).

  They exist as inactive precursors, the procaspase that is activated

by cleavage of aspartic acid by other caspases.

  Once activated caspases cleave and activates other procaspase in

a proteolytic cascade amplification (which is destructive, self-
driven and irreversible)

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 Apoptosis
  All nucleated animal cells contain several inactive
procaspases (only activated when there is need for
destruction of the cell).

  The activation of caspases cascade is triggered by adapter

proteins:
  Make multiple copies of specific procaspases (initiator
procaspases) join in a complex and cleave triggering a mutual
activation. It can also occur a conformational change that
activates the procaspases.

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Apoptosis

  The activation of procaspases can occur by:

  extrinsic pathway: activation of death receptors in cell surface.

  intrinsic pathway: the mitochondria to release cytosolic cytochrome C that

binds and activates the protein adapter Apaf-1.

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Apoptosis

  The initial activation of a small number of procaspases (initiator

caspases) leads to activation of a cascade with consequent activation of
multiple caspases. Some them (executing caspases) cleave key proteins
of cells (cytosolic and nuclear lamins).

Apoptosis

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Apoptosis

This pathway of apoptosis activation

requires the p53 protein that activates
gene transcription encoders proteins
that promote the release of cytochrome
c from mitochondria (family of proteins
Bcl-2).

Apoptosis Intracellular regulators

  Bcl-2 family proteins
  IAP family proteins (inhibitors
apoptosis)

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Apoptosis Intracellular regulators
  Bcl-2 family proteins: intracellular proteins
that regulate the activation of procaspases.
  Bcl-2, Bcl-XL: block apoptosis by inhibit the
release of cytochrome c from mitochondria.
  Bad: bind and inhibit the proteins that block
apoptosis.
  Bax, Bak: stimulate the release of cytochrome C
  Bid: activates Bax and Bak

Apoptosis Intracellular regulators

  IAP family proteins: Inhibit
apoptosis.
  Bind to procaspases preventing their
activation.
  Bind to caspases inhibiting its activity.

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 Cell Death Receptors and
Caspase Activation
  Some secreted polypeptides signal programmed cell death by
activating receptors that directly induce apoptosis of the
target cell.
  These cell death signals are polypeptides belonging to the
tumor necrosis factor (TNF) family.
  They bind to members of the TNF receptor family, which can
signal apoptosis in a variety of cell types.
  One of the best characterized members of this family is the cell
surface receptor called Fas, which plays important roles in
controlling cell death in the immune system.

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Cell Death Receptors

and Caspase Activation

  Binding of ligand to the Fas receptor

induces apoptosis by direct activation of
caspase-8.
  Fas ligand consists of three polypeptide
chains, so its binding induces receptor
trimerization.
  Caspase-8 bound to the receptor via
adaptor molecules is then activated by
autocleavage, leading to activation of
downstream caspases and cell death.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.49. 40

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Signaling Cell Survival

  Signaling by TNF and related polypeptides is an active

process in which stimulation of cell death receptors
induces apoptosis.
  Other signaling pathways act in the opposite direction
to promote cell survival by inhibiting apoptosis.

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Signaling Cell Survival

  One of the major intracellular signaling pathways responsible

for promoting cell survival is initiated by the enzyme PI 3-
kinase,
  which is activated by either protein-tyrosine kinases or G protein-
coupled receptors.

  PI 3-kinase phosphorylates the membrane phospholipid PIP2

to form PIP3,
  which activates the protein-serine/threonine kinase Akt.

  Akt then phosphorylates a number of proteins that

regulate apoptosis

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Signaling Cell Survival
Survival factors such as NGF activate receptor protein-
tyrosine kinases, leading to activation of PI 3-kinase and
formation of PIP3. PIP3 recruits the protein kinase Akt to
the plasma membrane where it is activated as a result of
phosphorylation by PDK.

Akt then appears to phosphorylate a number of proteins that contribute to

cell survival. The targets of Akt that have been implicated in suppression of
apoptosis include the Bcl-2 family member Bad, caspase-9, several
transcription factors, and the protein kinase GSK-3, which affects cell
metabolism and protein synthesis.

The Cell: A Molecular Approach. 2nd edition. Cooper GM. Sinauer Associates. Figure 13.50. 43

Feedback and Crosstalk

  It is important for you to not think of these pathway as linear
pathways that occur within a vacuum.
  It is important to understand that they occur within the cytoplasm
with numerous other proteins and pathways.
  Signaling pathways do not operate in isolation; rather, there is
frequent crosstalk between different pathways, so that intracellular
signal transduction ultimately needs to be understood as an
integrated network of connected pathways.
  In addition often the activities of individual pathways are regulated
by feedback loops that control the extent and duration of signaling
activity.
  Very often the activity of signaling pathways is regulated by
feedback loops which are similar in principal to the feedback
regulation often observed in metabolic pathways.

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 Feedback and Crosstalk
  Crosstalk refers to the interaction of one signaling pathway with
another.
  Very little that occurs within a cell occurs in a linear fashion. All
pathways have the ability to be regulated by other molecules or
signaling pathways that are present within the cytoplasm.
  Generally cell signaling starts at the plasma membrane and occurs
primarily within the cytoplasm. Sometimes what occurs in the
cytoplasm leads to the transport of transcription factors across the
nuclear membrane to interact with genes and ultimately activate
gene expression.

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Feedback and Crosstalk

  β-arrestins are
regulatory proteins that
terminate signaling
from G protein-coupled
receptors, as well as
stimulate other
downstream signaling
pathways.

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 G-protein coupled receptors and the ERK
signaling pathway by the molecule ß-arrestin.

Ligand binding
stimulates G-
protein coupled
receptors
which leads to
the activation
of trimeric G-
proteins.
The activity of the receptor is turned off as a result of
phosphorylation by GRKs and associatin with ß-arrestin
with the phosphorylated receptor.

ß-arrestin also acts as a scaffold protein for Raf, Mek, and Erk which links G-protein coupled
receptors to the Erk signaling pathway.

Networks of Cellular Signal

Transduction
  The extensive crosstalk between individual signal transduction

pathways means that multiple pathways interact with one

another to form signaling networks within the cell.

  Signaling networks are interconnected networks formed by the

interactions of multiple signaling pathways within a cell.

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 Networks of Cellular Signal
Transduction
  In feedback loops a downstream element of the pathway either inhibits
(negative feedback) or stimulate (positive feedback) an upstream element.
  In feedforward relays and upstream element of a pathway stimulates both its
immediate target and another element further downstream.
  Crosstalk occurs when an element of one pathway either stimulates or
inhibits an element of a second pathway.

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Networks of Cellular Signal

Transduction
  In order to fully understand the signaling that occurs within a cell, will
require the development of a network of models that predict the dynamic
behavior of the interconnected signaling pathways that ultimately result
in a biological response.
  In this view of cell signaling as an integrated system , mathematical
models and computer simulations will clearly be needed to deal with the
complexity of the problem.
  Think about the logistics of the human genome. It encodes about 1500
different receptors, almost 700 protein kinases and phosphatases, and nearly
2000 TFs, so the potential for cross regulation between pathways formed from
combinations of these elements is enormous…

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