LAB EVALUATION OF
Ola H. Elgaddar
MD, PhD, CPHQ, LSSGB
Lecturer of Chemical Pathology
Medical Research Institute
- There are large numbers of intracellular
signaling pathways responsible for transmitting
information within the cell.
They fall into two main categories:
The majority respond to external stimuli
arriving at the cell surface
Pathways that are activated by signals
generated from within the cell
- All of these signalling pathways generate an
internal messenger that is responsible for
relaying information to the sensors that then
engage the effectors that activate cellular
- The names of these signalling pathways usually
reflect a major component(s) of the pathway
1. Cyclic AMP signaling pathway
The idea is that the external stimulus arriving at
the cell surface is the first messenger, which is
then transformed at the cell surface by adenylyl
cyclase (AC) into a second messenger, cyclic
AMP, which is a part of the signaling cascade
that then activates down-stream effectors.
(AC converts ATP into cAMP)
- Many of the actions of cyclic AMP
are carried out by protein kinase A
(PKA), which phosphorylates
specific sites on downstream effector
- The cyclic AMP signalling pathway
functions in the control of a wide
range of cellular processes, like
oocyte maturation, heat production in
brown fat & glycogenolysis in
2. Cyclic ADP-ribose (cADPR) and nicotinic
acid–adenine dinucleotide phosphate (NAADP)
- The enzyme ADP-ribosyl cyclase is a bifunctional
enzyme that has a synthase (S) component that
synthesizes cADPR and NAADP from the
precursors NAD+ and NADP respectively, but it
also has a hydrolase (H) activity that converts
cADPR into ADPR.
- This hydrolase is sensitive to metabolism because
it is inhibited by either ATP or NADH.
- The cADPR may act by stimulating
the sarco/endo-plasmic reticulum
Ca2+-ATPase (SERCA) pump to
increase the uptake of Ca2+ into the
- NAADP acts on a channel to
release Ca2+ from a lysosome related
- Both cADPR and NAADP have rule
in insulin secretion by pancreatic β-
3. Voltage-operated channels (VOCs)
-Contribute to Ca2+ signals by controlling the entry
of external Ca2+ in excitable cells
- At resting membrane potential, the channels are
normally closed. They are activated (i.e., opened) at
depolarized membrane potentials.
- Activation of particular channel allows Ca2+ entry
into the cell resulting in muscular contraction or
excitation of neurons.
4. Receptor-operated channels (ROCs)
- They are a group of transmembrane ion channels
that are opened or closed in response to the binding
of a chemical messenger (i.e., a ligand), such as a
- Contribute to Ca2+ signals by controlling Ca2+
entry in both excitable and non-excitable cells.
5. Stimuli activating phospholipase C
(PLC) to hydrolyse PtdIns 4,5P2 (PIP2)
Generate a number of signaling pathways:
Inositol 1,4,5-trisphosphate (InsP3)/Ca2+
Diacylglycerol (DAG)/protein kinaseC (PKC)
6. PtdIns 3-kinase signaling
- Is activated by stimuli that stimulate PtdIns 3-
kinase to phosphorylate PIP2 to form the lipid
second messenger PtdIns3,4,5P3 (PIP3).
7. Nitric oxide (NO) signaling pathway
- Nitric oxide (NO) is a highly diffusible messenger,
which passes rapidly through cell membranes.
- It can act as a second messenger within its cell of
origin or it can diffuse across membranes to act on
neighboring cells as a paracrine signaling agent.
- NO synthesis is carried out by nitric oxide
NO actions are mediated in two ways:
Through the cyclic GMP signaling
where it stimulates soluble guanylyl
cyclase to produce the cyclic GMP
that can modify the properties of Ca+2
channels to promote Ca2+ entry or it
can activate cyclic GMP-dependent
Through the reactive nitrogen
species (RNS) signaling pathways
- whereby the NO alters the activity of
a variety of protein targets through a
NO/cyclic GMP signaling pathway
operates to control many cellular
processes such as smooth muscle
relaxation, platelet inhibition and
changes in gene expression.
8. Redox signaling
- Cells have evolved a sophisticated mechanism of
intracellular signaling based on localized changes in
the oxidation state of specific proteins.
- The internal environment of cells is normally
- Certain forms of stress are associated with an
increase in the oxidative state.
Two main types of redox signaling
Reactive oxygen species (ROS) signaling
Reactive nitrogen species (RNS) signaling,
which is carried out by RNS and is linked to the
nitric oxide (NO)/cyclic GMP signaling pathway.
9. Mitogen-activated protein kinase
-MAP kinases are serine/threonine-specific protein
kinases that respond to extracellular stimuli (mitogens,
osmotic stress, heat shock and pro-inflammatory
cytokines) and regulate various cellular activities, such
as gene expression, mitosis, differentiation,
proliferation, and cell survival/apoptosis.
-MAP kinases are activated within the protein kinase
cascades called MAPK cascade or MAPK signaling
- Each one consists of three enzymes, MAP kinase,
MAP kinase kinase (MKK, MEK, or MAP2K) and
MAP kinase kinase kinase (MKKK, MEKK or
MAP3K) that are activated in series.
- MAP3K that is activated by extracellular stimuli
phosphorylates a MAP2K on its serine and threonine
residues, and this MAP2K activates a MAP kinase
through phosphorylation on its serine and tyrosine
10. Nuclear factor κB (NF-κB) signaling
-The transcription factor nuclear factor κB (NF-κB) is
activated by a large number of external stimuli such as
the tumour necrosis factors (TNFs) and interleukin-1
(IL-1), which are responsible for controlling processes
such as inflammation, cell proliferation and apoptosis.
- NF-κB belongs to the group of transcription factors
that lie latent in the cytoplasm and then translocate into
the nucleus upon activation.
NF-κB resides in the cytosol bound to an inhibitor called IκB.
Binding of ligand to the receptor triggers phosphorylation of IκB
IκB then becomes ubiquinated (added to Ubiquitin protein) and
destroyed by proteasomes.
This liberates NF-κB so that it is now free to move into the
It acts as a transcription factor binding to the promoters and/or
enhancers of many genes
11. Phospholipase D (PLD) signaling
- PLD is an enzyme that hydrolyzes the
phosphodiester bond in phosphatidylcholine (PC),
yielding choline and phosphatidic acid (PA)
- PA is the primary messenger in PLD signaling
pathway, which acts to regulate a wide range of
cellular processes, including cytoskeletal
rearrangement, vesicle trafficking, exocytosis,
phagocytosis, oncogenesis, and neuronal and cardiac
- Phosphatidic acid (PA) action is carried out
through a number of downstream effectors such as
protein kinase C, G-proteins, PIP3 and GTP.
- The action of PA is terminated either by a PA
phosphohydrolase, which removes phosphate to
leave behind diacylglycerol (DAG), or by a
phospholipase A2 (PLA2) to produce
lysophosphatidic acid (LPA).
12. Sphingomyelin signaling pathway
- Certain growth factors and cytokines hydrolyse
sphingomyelin to generate two secondary messengers that
appear to have opposing effects in the cell.
- Ceramide seems to promote apoptosis, whereas
sphingosine 1-phosphate (S1P) stimulates cell
- The action of S1P is complicated in that it is released
from the cell, where it can act as a hormone to stimulate
13. Janus kinase (JAK)/signal transducer and
activator of transcription (STAT) signaling
- The Janus kinases (JAKs) are tyrosine kinases that
phosphorylate the signal transducers and activators of
transcription (STATs), which are latent transcription
- Once phosphorylated, these STATs leave the membrane
and then dimerize before migrating into the nucleus
where they bind to specific DNA-binding elements to
This signaling pathway is mainly activated by
cytokines such as interferon, but is also used by
receptor tyrosine kinases [epidermal growth
factor receptor (EGFR), platelet-derived growth
factor receptor (PDGFR)], nonreceptor tyrosine
kinases and G-protein-coupled receptors
14. Smad signaling pathway
- This signaling pathway takes its name from the Smads,
which are a collection of intracellular signaling molecules
that act collectively to transfer information from cell
surface receptors into the nucleus.
- As such, some of the Smads function as transcription
factors, whereas others either facilitate or inhibit this
- These Smads mediate the action of the transforming
growth factorβ (TGF-β) superfamily
-The Smad signaling mechanism can be divided into
two parts. Firstly, there is the process of TGF-β
receptor activation, which concerns the way in which
ligands (TGF-β) interact with the signaling receptors.
-The critical aspects of this activation process are the
phosphorylation reactions that occur within the receptor
complex. The Type II receptors are constitutively active
and phosphorylate the Type I receptors. These activated
Type I receptors then act to phosphorylate the Smads.
The second part is the Smad activation of
transcription, during which the phosphorylated
receptor-regulated Smads (R-Smads), together
with their partner Smad4, translocate into the
nucleus to induce gene transcription
15. Wnt signaling pathways
- The Wnt signalling pathways play a critical role in the
control of cell proliferation and differentiation.
- These different pathways are activated by extracellular
lipoprotein signaling molecules called Wnts, responsible
for transmitting information between cells over relatively
- This pathway is involved in the development of many
diseases such as obesity, early coronary diseases,
Alzheimer and Polyposis coli.
16. Hedgehog signaling pathway
- Hedgehog mediates its effects by activating gene
- It gives cells information that they need to make the
embryo develop properly. Different parts of the embryo
have different concentrations of hedgehog signaling
- There are three Hedgehog transcription factors (GLI 1–3),
which are held in an inactive state within the cytoplasm in
- This inactive state is maintained by the Hedgehog
receptor patched (PTC), which inhibits the seven
membrane- spanning protein smoothened (SMO)
that acts as the Hedgehog transducer.
- In the absence of a signal from SMO, theGLI
transcription factors are maintained in a latent state
by interacting with a large number of cytoplasmic
- Hedgehog arriving at the cell surface induces a
cascade of events that activate these transcription
factors so that they translocate into the nucleus to
induce gene transcription.
- First of all, Hedgehog binds to its receptor PTC
and this removes the inhibitory effect of PTC on
SMO. The latter is then able to activate GLI by
removing it from the inhibitory constraints of the
cytoplasmic factors so that it is now free to
translocate into the nucleus to activate
17. Notch signaling pathway
- This is a short-range information transfer mechanism that
depends upon direct contact between the cells, which is a
hallmark of this signaling pathway.
- For example, the stimuli Jagged or delta are integral
membrane proteins located on the surface of communicating
cells, whereas the Notch receptor that responds to them is
embedded in the surface of the receiving cell.
- When Delta or Jagged interacts with Notch (through
their [DSL] = Delta, Serrate and LAG-2 domain), this
triggers a series of steps that result in the proteolytic
release of the Notch intracellular domain (NICD),
which then enters the nucleus to activate transcription of
Notch target genes.
- Notch pathway is involved in many functions
including, neuronal development, angiogenesis, cardiac
valve homeostasis and bone formation.
18. Endoplasmic reticulum (ER) stress signaling
- It is concerned with the mechanisms used by the ER to
transmit information to the nucleus about the state of protein
processing within the lumen of the ER.
- Maintenance of a constant level of Ca2+ within the lumen of
the ER is essential for the post translational processing,
folding and export of proteins.
- This protein processing is carried out by a number of Ca2+-
- Any decline in the luminal level of Ca2+
results in the accumulation of misfolded
proteins and the activation of the ER stress
- Oligomerization and autophosphorylation
of PKR (protein kinase R)-like ER
kinase (PERK) sets off a phosphorylation
cascade resulting in protein synthesis being
- ER stress pathway contributes to
apoptosis through activation of caspase 12
and transcription factor nuclear factor κB
19. AMP signaling pathway
- Cells have an AMP signaling pathway that is activated by
an increase in the AMP/ATP ratio, which results in the
activation of an AMP-activated protein kinase (AMPK)
- AMP thus functions as a second messenger, since it is
responsible for activating the signaling pathway.
- The AMPK that responds to AMP has been referred to as
the “fuel gauge” in that it responds to a decrease in the level
- This signaling cascade is sensitive to many
stimuli, such as cell stress, oxidative damage,
hypoxia and glucose deprivation.
- Once activated, AMPK induces an up-
regulation of ATP-generating systems while
simultaneously down-regulating processes that
consume energy, such as reducing protein