3-1) Lecture 21 - Signal Transduction I

Signal Transduction

the process by which an extracellular signal is amplified and converted to a cellular response.

What are examples of extracellular signals?

Molecular signals from other cells, environmental stresses, extracellular matrix, nutrient signals

What are examples of cellular responses?

Gene expression, morphological changes, production/secretion, cell-growth/division, cell death.

What are the three steps of signal transduction?

1) Reception (receptors sense chemical signals),

2) Transduction (signals are recoded, amplified, and transmitted through a signal transduction pathway consisting of discrete chemical reactions),

3) Response (cellular targets bring about responses underscored by chemical changes)

How to mediate an on-off switch of a PROTEIN in signal transduction?

Activation or inactivation of signaling enzymes

change in binding interaction between proteins or between proteins and other biomolecules

change in protein conformation,

post-translational modification,

change in subcellular localization or local concentration

How to mediate an on-off switch of a NON-PROTEIN SMALL MOLECULE in signal transduction?

Production of intracellular small signaling molecules (second messengers).

What is the role of enzymes in signal transduction?

Catalyze chemical reactions which control many key steps in signaling,

write and erase signals in a controlled fashion,

provide simple mechanisms for signal amplification.

What are the major classes of signaling enzymes?

Covalent modification of proteins and lipids (kinases, phosphatases / proteases, lipases),

Effecting conformational change in other proteins (Guanine nucleotide exchange factor (GEF),

GTPase-activating protein (GAP)),

Generation and elimination of small signaling molecules (Adenylyl cyclase, phosphodiesterase)

Protein kinase

catalyze protein phosphorylation by transferring -phosphate of ATP to the side chain of selected amino acids.

They can be soluble proteins or membrane proteins (the kinase domain exists as an intracellular domain).

Function as molecular switches because they can exist in active and inactive conformational states.

In eukaryotes, protein phosphorylation occurs on

the hydroxyl group of serine, threonine (Ser/Thr protein kinases) and tyrosine (Tyr protein kinases).

Structure of a eukaryotic protein kinase catalytic domain (kinase domain)

a two-lobed structure, the active site (catalytic), and activation loop (regulatory)

How does upstream input signal control protein kinase activity?

Through the binding of an activator protein or release of an inhibitor protein,

through the production of a small molecule allosteric regulator,

through the phosphorylation of an activation loop of the protein kinase.

Kinase cascade

activation of an upstream protein kinase leads to the phosphorylation of a downstream protein kinase. (think cascade, waterfall, goes from upstream to downstream)

How does protein phosphorylation work?

It is a type of post-translational modification that changes the chemical property of the modified amino acid.

Changes a polar uncharged amino acid to a negatively charged amino acid, changing the isoelectric point (pI) of the modified protein.

What are the signaling outputs of protein phosphorylation?

Change in protein conformation and activity,

promote or prevent protein binding interaction,

change protein subcellular localization,

change in protein stability.

Protein phosphatase

an "eraser" of protein phosphorylation (think of tasing a phosphate off the protein). Its activity is subjected to regulation by upstream input signals.

The kinase and phosphatase reaction forms a...

unidirectional cycle. Both reactions steps can be controlled and regulated.

What are the three phosphatase subfamilies (soluble or membrane bound)?

Serine/threonine phosphatases

Tyrosine phosphatases

Dual specificity phosphatases

Guanine nucleotide-binding proteins (G proteins)

bind and hydrolyze GTP, function as molecular switches in a wide variety of biological processes.

Small G proteins

monomeric (about 150 in humans), consists of a single G domain

Heterotrimeric G proteins

Have α, β, and γ subunits

(~16 α subunit in humans.)

It consists of a G domain in its α subunit. The β subunit and the γ subunits form a stable complex and have no enzymatic activity.

How is the heterotrimeric G protein an integral component in the G protein couple receptor (GPCR) signaling pathway

In the INACTIVE form, Gα is bound to GDP and associates with Gβγ to form a hetero-trimer.

In the ACTIVE form, Gα is bound to GTP and dissociates from Gβγ, which can both interact with downstream effectors.

All G proteins contain the ___ domain, which binds GTP.

20kDa G

How does G protein mediate signal transduction?

Through the binding and hydrolyzing of GTP.

GTP-bound = on, GDP-bound = off.

When GTP is bound, the terminal γ-phosphate interacts with switch I and II, causing a conformational rearrangement to form a critical effector binding site for the G protein to bind to an effector.

Residues of the G protein outside of switch I and II determine which specific effector it binds.

How are G protein-mediated signal transduction turned on and off?

GEFs or Guanine nucleotide exchange factors accelerate GDP/GTP exchange and therefore turn on G proteins.

GTPase-activator proteins (GAPs) accelerate GTP hydrolysis and inactivate G proteins.

From off to on exchange of GDP with GTP is very slow, and from on to off G proteins are poor GTPases.

Biological SIGNALING processes that are impacted by G proteins

hormonal action, cytoskeletal regulation, nuclear import and export

Biological NON-SIGNALING processes that are impacted by G proteins

translation, translocation, vesicle trafficking, etc.

Guanine nucleotide exchange factor (GEF)

Accelerate GDP/GTP exchange thus activating G proteins.

GTPase-activator protein (GAP)

accelerates GTP hydrolysis thus inactivating G proteins.

Second messenger

intracellular small signaling molecules

IP3-gated Ca2+-channel

Channel targeted by IP3 (inositol trisphosphate) in the endoplasmic reticulum.

Ca2+-ATPase pump

Removes systolic Ca+2 in the absence of hormonal, neural, or other signals.

Calmodulin (CaM)

common conformational sensor of intracellular Ca+2.

Ca+2 binding regulatory protein (CaM). Undergoes conformational change when Ca+2 binds, it associates with a variety of prteins and modulates their activities in a Ca+2 dependent manner.

Is a regulatory subunit of phosphorylase b kinase of muscle which is activated by Ca+2.

Modular domain

signaling proteins that are frequently composed of multiple domains, joined together by flexible linker sequences.

Domains within larger proteins that retain their distinct structure and activity.

2 types: (catalytic and interaction domains)

Src Homology 2 (SH2) domain

Recognizes post-translational modification

binds phospho-Tyr containing sequence.

Recognition of phospho-Tyr is achieved through the conserved phospho-tyrosine binding pocket.

Different ___ domains show specific preference for residues, C-terminal to phospho-tyrosine (achieved through the "specificity pocket".

How do second messenger molecules mediate signal transduction?

Control intracellular targets affecting processes that bring about the response to the extracellular signal.

It acts as an allosteric regulator of downstream effectors (e.g. enzymes and channels).

Lipid-derived molecules can affect membrane recruitment of target proteins.

Can lead to fast, distant and amplified signal transmission. 

How are second messengers controlled?

By their production and elimination.

Activation leads to increased synthesis of the signalling molecules (sometimes decreased degradation.

The concentration of Ca2+ is regulated by the opposing activities of the pumps and channels.

cAMP (adenosine 3’, 5’-cyclic monophosphate)

a soluble second messenger, synthesized from ATP, catalyzed by adenylyl cyclase.

Diacylglycerol (DAG)

synthesized from PI(4,5)P2, catalyzed by phosphoinositide-specific phospholipase C.

It’s a membrane lipid, stays within the membrane to bind and activate protein kinase C (PKC) along with Ca2+.

Inactivated by phosphorylation which makes phosphatidic acid. 

Inositol triphosphate (IP3)

synthesized from PI(4,5)P2, catalyzed by phosphoinositide-specific phospholipase C.

it’s water soluble and diffuses rapidly through the cytoplasm.

Its target is an IP3-gated Ca2+ channel in the endoplasmic reticulum.

Inactivated by desphosphorylation

IP3-gated Ca2+-channel

Channel targeted by IP3 (inositol trisphosphate) in the endoplasmic reticulum.

How is intracellular calcium ion concentration controlled?

By channels and pumps.

In the PRESENCE of hormonal, neural, or other signals, cystosolic [Ca2+] rises through an influx of extracellular Ca2+ through gated-Ca2+ channel and the release of Ca2+ from internal storage through gated-Ca2+ channel.

In the ABSENCE of signal, cytosolic Ca2+ is removed by Ca2+-ATPase pumps.

Ca2+ signaling tends to be ___ and ___ because there is a high concentration of Ca2+ binding proteins in the cytoplasm

Transient and local.

Ca2+-ATPase pump

Removes systolic Ca2+ in the absence of hormonal, neural, or other signals.

Effectors of Ca2+ signaling generally DO NOT bind to Ca2+ directly, but are regulated by…

a common Ca2+-binding regulatory protein, CaM,

Phosphatidylinositol phosphates (PIPs)

formed by the phosphorylation of the 3, 4, 5-OHs of phosphatidylinositol (PI). Different PIPs are recognized by specific phospholipid-binding protein domains.

How are phosphatidylinositols (PIs) phosphorylated and dephosphorylated?

Phosphorylated by various PI kinases. Dephosphorylated by phosphatidylinositol phosphatases.

Modular domain

signaling proteins that are frequently composed of multiple domains, joined together by flexible linker sequences.

Domains within larger proteins that retain their distinct structure and activity.

2 types: (catalytic and interaction domains).

What are other modular domain that recognize post-translational modification marks?

PTB domain (phospho-Tyr)

14-3-3 domain (phospho-Ser/Thr)

bromodomain (acetyl-Lys), chromodomain (methyl-Lys).

Src Homology 3 (SH3) domain

Recognizes unmodified peptide motifs or proteins) binds short, proline-rich sequence (pxxP) motifs.

Proline-rich sequences favor the formation of polyproline type 2 (PPll) helix, a left-handed helix with three residues per turn.

The binding pocket strongly disfavors a polar amide group while tolerates a more hydrophobic N substitution (proline) 

What are other modular domains that recognize unique sequence or structural features?

EVH1 domain (proline rich sequence),

PDZ domain (protein C-terminus),

SAM domain (protein polymer)

Pleckstrin homology (PH) domain

Recognizes phospholipids) binds the phosphorylated inositol head group of PI(4,5)P2 and PI(3,4,5)P3.

Specificity of Phosphoinositide recognition is determined by the subtle differences in the binding pocket.

What are other phosphoinositide-binding domain

FIVE domain [PI(3)P],

PX domain [PI(3)P],

CALM domain [PI(4,5)P2]

What is the advantage of modular domains?

Complex signal transduction scheme can be created by combining different domains.

Complex allosteric regulatory mechanisms can be devised for signaling enzymes.

Domain recombination can also generate proteins with new functions.

Adaptor protein

Proteins with multiple protein-binding domains that mediate the assembly of complexes of three or more components.

Scaffold protein

Proteins containing multiple protein-binding domains that can be used to organize complex signaling pathways.

They promote efficient signaling between component proteins and insulate them from cross-talk with incorrect but potentially competing partner proteins.

Guanine nucleotide exchange factor (GEF)

regulator of G protein, it’s an upstream input signal that controls G protein activity. Accelerates GDP/GTP exchange. ACTIVATES G proteins. 

GTPase-activator protein (GAP)

regulator of G protein, it’s an upstream input signal that controls G protein activity. Accelerates GTP hydrolysis. INACTIVATES G proteins.

Adenylyl cyclase

class of signaling enzyme that generates and eliminates small signaling molecules. Catalyzes reaction of synthesizing cAMP (soluble second messenger) from ATP.

Cyclic nucleotide phosphodiesterase (PDE)

hydrolyzes cAMP to AMP, eliminating its signaling function

cAMP-dependent protein kinase (PKA)

the main intracellular target of cAMP. Binding of cAMP to PKA activates the enzyme.

Phospholipase C (PLC)

Catalyzes the synthesis of DAG and IP3 from PI(4,5)P2.

Protein kinase C (PKC)

Located in membrane, activated by DAG (membrane lipid)

Phosphoinositide 3-kinase (PI-3 kinase)

phosphorylate the 3-OH of PI, PI(4)P, and PI(4,5)P2 to generate PI(3)P, PI(3,4)P2 and PI(3,4,5)P3.

PTEN

A phosphatase that removes the 3-phosphate from PI(3,4,5)P3. Also a tumor suppressor.

Adenosine 3’,5’-cyclic monophosphate (cAMP) (slide 20)

Phosphatidylinositol 4,5-bisphosphate (PIP2) (slide 21)

Inositol 1,4,5-trisphosphate (slide 21)

Diacylglycerol (slide 21)

Phosphatidylinositol 3,4,5-trisphosphate (PIP3) (slide 24)