LAB EVALUATION OF CELL DISORDERS

4) Signaling

Ola H. Elgaddar MD, PhD, CPHQ, LSSGB

Lecturer of Chemical Pathology

Medical Research Institute

Alexandria University

Ola.elgaddar@alexu.edu.eg

- 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

responses

- 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

processes.

- 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

skeletal muscles.

2. Cyclic ADP-ribose (cADPR) and nicotinic

acid–adenine dinucleotide phosphate (NAADP)

signaling systems

- 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

endoplasmic reticulum.

- NAADP acts on a channel to

release Ca2+ from a lysosome related

organelle.

- Both cADPR and NAADP have rule

in insulin secretion by pancreatic β-

cells.

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

neurotransmitter.

- 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+

signaling cassette

Diacylglycerol (DAG)/protein kinaseC (PKC)

signaling cassette

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

synthase (NOS)

NO actions are mediated in two ways:

Through the cyclic GMP signaling

pathway,

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

protein kinase

Through the reactive nitrogen

species (RNS) signaling pathways

- whereby the NO alters the activity of

a variety of protein targets through a

nitrosylation reaction.

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

highly reduced.

- 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

(MAPK) signaling

-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

toolkit

- 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

residues.

10. Nuclear factor κB (NF-κB) signaling

pathway

-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.

Mechanism:

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

nucleus where

It acts as a transcription factor binding to the promoters and/or

enhancers of many genes

11. Phospholipase D (PLD) signaling

pathway

- 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

stimulation

- 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

proliferation.

- The action of S1P is complicated in that it is released

from the cell, where it can act as a hormone to stimulate

external receptors.

13. Janus kinase (JAK)/signal transducer and

activator of transcription (STAT) signaling

pathway

- The Janus kinases (JAKs) are tyrosine kinases that

phosphorylate the signal transducers and activators of

transcription (STATs), which are latent transcription

factors.

- Once phosphorylated, these STATs leave the membrane

and then dimerize before migrating into the nucleus

where they bind to specific DNA-binding elements to

activate transcription.

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

transcriptional activity.

- 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

short distances.

- 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

transcription.

- It gives cells information that they need to make the

embryo develop properly. Different parts of the embryo

have different concentrations of hedgehog signaling

proteins.

- There are three Hedgehog transcription factors (GLI 1–3),

which are held in an inactive state within the cytoplasm in

resting cells.

- 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

factors

- 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

transcription

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+-

sensitive chaperones.

- Any decline in the luminal level of Ca2+

results in the accumulation of misfolded

proteins and the activation of the ER stress

signaling pathways.

- Oligomerization and autophosphorylation

of PKR (protein kinase R)-like ER

kinase (PERK) sets off a phosphorylation

cascade resulting in protein synthesis being

switched off.

- ER stress pathway contributes to

apoptosis through activation of caspase 12

and transcription factor nuclear factor κB

(NF-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

of ATP.

- 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

synthesis.

THANK YOU