lec 21 signal transduction I

signaling enzymes, second messengers, modular domains

signal transduction

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

extracellular signals

• molecular signals from other cells

• environmental stresses

• extracellular matrix

• nutrient signals

cellular responses

• gene expression

• morphological changes

• production/secretion

• cell growth/division

• cell death

signal transduction chemical process steps

reception

transduction

response

signal reception

most signals are chemical and are first sensed by receptors

signal transduction

once signals are detected, they will be recoded, amplified, and transmitted through a signal transduction pathway consisting of discrete chemical reactions

signal response

when acted upon by the relay molecules, cellular targets bring about responses underscored by chemical changes

a limited number of mechanisms mediate signal transduction

proteins and non-protein small molecule

proteins as a on-off switch mechanism

• 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

non-protein small molecule as a on-off switch mechanism

production of intracellular small signaling molecules (second messengers)

part 1: signaling enzymes

roles of enzymes in signal transduction

• catalyze chemical reactions which control many key steps in signaling (hella convenient)

• write and erase signals in a controlled fashion

• provide simple mechanisms for signal amplification

major classes of signaling enzymes

• covalent modification of proteins and lipids

• effecting conformational change in other proteins

• generation and elimination of small signaling molecules

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

enzyme catalytic activity can be controlled through input-driven protein conformational change

protein kinases

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

in eukaryotic cells, protein phosphorylation occurs on…

• the hydroxyl group of serine, threonine (Ser/Thr protein kinases)

• hydroxyl group of tyrosine (Tyr protein kinases)

protein kinases can be…

• soluble proteins (floating in cytosol)

• membrane proteins (the kinase domain exists as an intracellular domain)

  • can be localized in the membranes

  • typically consist of integral transmembrane segments, but could also have a domain hanging out from the membranes

structure of eukaryotic protein kinase catalytic domain

• despite the variety in protein kinases, they all have a catalytic domain that has a common overall structure:

  • two-lobed structure

    • N-lobe (n terminal)

    • inbetween lobes is the catalytic site

    • C-lobe (c terminal)

  • active site (catalytic)

  • activation loop (regulatory)

protein kinases function as molecular switches because they can exist in two conformational states: active and inactive

active = protein phosphorylation

inactive = no protein phosphorylation

upstream signals can use a variety of mechanisms to help regulate the equilibrium between the active and inactive conformation → leads to signal transduction

upstream signals activate the kinase → signal propagations

three major mechanisms for regulating protein kinase activity

1. binding of an activator protein or release of an inhibitor protein

2. through the production of a small molecule allosteric regulator (ex. second messenger)

3. through phosphorylation on the activation loop of the protein kinase

kinase cascade

activation of an upstream protein kinase leads to the phosphorylation and activation of a downstream protein kinase

protein phosphorylation changes the chemical property of the modified amino acid

• from a polar uncharged amino acid to a neg charged amino acid

• change the pI (isoelectric point) of modified protein

  • (more phosphorylation = more significant change in pI)

phosphorylation leads to these signaling outputs

• change in protein conformation and activity

• promote or prevent protein binding interaction

• change protein subcellular localization

• change protein stability (less or more stable)

protein phosphatase

“eraser” of the protein phosphorylation; counteracts the protein kinase

protein phosphatase subfamilies

• serine/threonine phosphatases

• tyrosine phosphatases

• dual specificity phosphatase

protein phosphatase activity is also subject to regulation by upstream input signal

kinase and phosphatase reaction form a unidirectional cycle in which both reaction steps can be controlled and regulated independently because they are independent rxns

true statements about protein phosphorylation in eukaryotic cells

• it can be reversed by the action of a protein phosphatase

• it is catalyzed by a protein kinase

• phosphoryl group usually derives from the gamma-phosphate of ATP

• happens on ser, thr, and tyr side chains only

G proteins (guanine nucleotide-binding proteins)

• also molecular switches

  • can exist in two diff states (on or off)

• bind and hydrolyze GTP

• when bound to GTP = ON

  • able to interact with downstream effector molecules

• when bound to GDP = OFF

G protein functions

• signaling: hormonal action, cytoskeletal regulation, and nuclear import and export

• non-signaling: translation, translocation, vesicle trafficking

why can G proteins go through conformational switch?

gamma-phosphate on GTP interacts with switch 1 and switch 2 → induces reaction and interactions cause significant conformational rearrangements in switch I and II → enables G protein to interact with downstream effective molecules

to go from off to on conformation in G protein

exchange of GDP with GTP; it is very slow, so it is paired with GEFs

GEFs aka guanine nucleotide exchange factors

• paired when G protein needs to go from off (GDP-bound) to on (GTP-bound)

• accelerates GDP/GTP exchange to activate G proteins

to go from on to off conformation in G protein

• GTP will undergo hydrolysis that is accelerated by GTPase-activator proteins (GAPs) because the reaction is also very slow if not without the catalytic enzyme

• leads to inactivation

able to control the molecular switch of G proteins through…

GEF and GAP b/c GEF activates and GAP inactivates

Two main families of G proteins that play key roles in cell signaling

• small G proteins: monomeric (only one subunit)

• heterotrimeric G proteins: have alpha, beta, and gamma subunits

All G proteins contain the…

20 kDa G domain which bines GTP

• small G protein = 1 G domain

• heterotrimeric G protein = G domain in alpha subunit

heterotrimeric G proteins

• plays role in G protein couple receptor (GPCR) signaling pathway

• its beta and gamma subunits form a stable complex and have no enzymatic activity

• beta-gamma is tightly associated therefore never dissociate

hetereotrimeric G protein in inactive form

G-alpha bound to GDP

beta-gamma of G forms hetero-trimer bc they associate with each other

heterotrimeric G protein in active form

G-alpha bound to GTP

G-alpha dissociates beta-gamma of G

both Gs can interact with downstream effectors

part 2 second messengers

second messenger

small signaling molecule that acts as an allosteric effector for a protein or an enzyme

bind to receptors and then receptors begin signal transaction pathway

• molecules that are generated in response to signal transduction inside the cells

properties of second messengers

• small

• signaling controlled by synthesis/production and elimination/degradation

second messengers exert their effects by acting as allosteric regulators of downstream effectors

• targets include a mide-range of enzymes and chennels

• lipid derived-molecules can affect membrain recruitment of target proteins

second messengers lead to fast, distant, and amplified signal transmission

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

• derived from ATP

• soluble second messenger

• catalyzed by adenylyl cyclase

cyclic nucleotide phosphodiesterase (degrader)

hydrolyzes cAMP to AMP → eliminating its signlaing function

what is the main intracellular target of cAMP

• protein kinase A (PKA)

  • downstream effector of cAMP

• binding of cAMP to PKA activates the enzyme

inositol triphosphate (IP3)

• synthesized from PI(4,5)P2

• catalyzed by phosphoinositide-specific phospholipase C

• water soluble therefore able to do stuff in the cytosol

• has gated calcium channel in ER ← when IP3 bounded to gated calcium channel, it opens up and Ca will be released

diacylglycerol (DAG)

• synthesized from PI(4,5)P2

• catalyzed by phosphoinostide-specific phospholipase C

• remains in membrane lipid

• binds and activiates protein kinase C (PKC)

PKC

activated by DAG and calcicum conentration

calcium ion

concentration regulated by channels and pumps (degradation and production)

• cytosolyc calcium concentration rises through in the presence of hormonal, neural, or other signals

  • an influx of extracellular calcium through gates calcium channel

  • the release of calcium from internal storage through gated calcium channel

absence of a signal = cytosylic calcium is removed by…

calcium-ATPase pumps

ca signaling is transient and local

bc high concentration of calcium in cells

effectors of calcium signaling regulated regulatory protein calmodulin (CaM)

• why? because effectors of Ca do not typically bind to Ca directly

calmodulin (CaM)

• contains four calcium binding sites

  • undergoes conformational change when Ca binds to it

    • when bound, hydrophobic surface is exposed which can then interact with downstream effector molecules

• regulatory subunit of phosphorylase of beta kinase of muscle, which is activated by calcium

phosphatidylinositol phosphates

• 3 4 5 hydroxyl groups of it can be phosphorated to form PIPs

  • diff PIPs are recognized by specific phospholipid-binding protein domains

phosphorylation of phosphatidylinositol is done by phosphatidylinositol (PI) kinases

PI-3 kinases phosphorylates the hydroxyl group on the 3 position

reverse reaction is catalyzed by phosphatidylinositol phosphatases

• dephosphorylates PIPs

• a phosphatase that removes 3-phosphate from PI (3,4,5) is a tumor suppressor called PTEN

overview of what produces (catalyzes) what second messenger

• adenylyl cyclase → cyclic AMP (cAMP)

• phospholipase C → diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP3)

• phosphatidylinositol-3 (PI-3) kinase → phosphatidylinositol types = PI3,4,5 and PI3 kinase

part 3 modular domains

modular domains

• signaling proteins are frequently composed of multiple domains, joined by flexible linker sequences (“loops)

  • the flexible linker sequences allows the domains to retain their unique structures and unique functions

total function of a signal in proteins is equal to

the linear summation of all of the structure functions of these individual domains

advantages 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

two types of modular domains

catalytic and interaction

first class of modular domain: domains that recognize post-translational modification

• ex. kinase (protein phosphorylation)

  • creates a new structure motif (new sites for protein-protein interaction) that can be recognized by other modular domains

• ex. Src Homology 2 (SH2) domain: binds phospho-Tyr containing sequence

  • creates recognition site by an SH2 domain

  • SH2 recognized protein contains SH2 domain

  • different SH2 domains show specific preference for residues C-terminal to phospho-tyrosine (achieved through specificity pocket)

second class of modular domain: domains that recognize UNMODIFIED peptide motifs or proteins

• protein conformational changes associated with signal transduction often expose unique sequence or structural features, which are recognized by modular protein interaction domains

• ex. Src Homology 3 (SH3) domain: binds short, proline-rich sequence (PxxP) motives

  • proline-rich sequences favor the formation of polyproline type II (PPII) helix, a left-handed helix with three residues per turn

    • contains binding pocket that disfavors amino acids that do not contain proline

      • disfavors polar amide group in backbone of amino acid (so all other 19 amino acids) but it can tolerate a more hydrophobic N substitution (proline)

Src Homology 3 (SH3) domain

proline-rich sequences favor the formation of polyproline type II (PPII) helix, a left-handed helix with three residues per turn

contains binding pocket that disfavors amino acids that do not contain proline

disfavors polar amide group in backbone of amino acid (so all other 19 amino acids) but it can tolerate a more hydrophobic N substitution / ring structure(proline)

three class of modular domains: domains that recognize phospholipids

• selectively bind phospholipids within biological membrane

  • target a protein to a particular subcellular organelle

  • localize a protein to a membrane

Pleckstrin homology (PH) domain

• binds the phosphorylated inositol head group of these second messenger molecules → PI (4,5)P2, and PI(3, 4, 5)P3

  • specificity of phosphoinositide recognition is determined by the subtle differences in binding pocket

• scenario: upstream signaling can lead to the activation of a kinase that can phosphorylate phosphatidylinositol molecules → lead to the binding of PH domain

  • phosphatidylinositol molecule will be recruited from the cytosol to the membrane because the lipids are actually membrane localized

adaptor protein

• multi-domain protein that consist of modular domains that only binds to other molecules

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

  • ex. Grb2 protein links phospho-tyrosine containing receptors to downstream targets that contain proline-rich sequences

    • in the absence of Grb2, the protein that contains the proline-rich sequences CANNOT interact with the protein containing phospho-tyrosine

    • in the presence of Grb2, the proline-rich protein & phospho-tyrosine protein can interact with each other

    • acts as an adaptor, like a circuit and an outlet needing an adaptor

scaffold protein

• multi-domain protein that consist of modular domains that only binds to other molecules

• function: to organize complex signaling pathways; bind a series of proteins in a given signal transduction pathways

  • binds all the components within the signal transduction pathway

• advantage: bring these components together and minimize the unproductive interaction among other proteins