Biosignaling

CH353

March 13–25, 2008

Summary

• Introduction to Biosignaling• Receptor-Ligand Specificity and Sensitivity• Signal Transduction Themes• Signaling with G Protein Coupled Receptors

– GPCRs stimulating or inhibiting adenylate cyclase

– GPCRs stimulating phospholipase C

– GPCRs involved in sensory reception

• Signaling with Receptor Enzymes– Receptors with guanylyl cyclases

– Receptors with intrinsic tyrosine kinase activity

– Receptors that recruit tyrosine kinases

• Regulation of Cell Cycle Protein Kinases

Types of Signaling

Endocrine signaling• Signaling molecules act on distant target cells

• hormones

Paracrine signaling• Signaling molecules act on nearby target cells

• neurotransmitters, growth factors, cytokines

Autocrine signaling• Signaling molecules act on originating cell

• tumor growth factors

Juxtacrine signaling• Attached signaling molecules act on adjacent

target cells

• integrins, cell adhesion molecules

Signal Transduction Pathways

Common Elements• Receptor mediated transfer of signal

inside of cell (mostly membrane receptors)

– formation of receptor-ligand complex

– most ligands remain outside cell

• Relay and amplification of signal from receptor-ligand complex– cascades of protein and enzyme

modifications and product synthesis

– GTPase switch proteins, kinases and phosphatases, second messengers

• Termination of signal– hydrolytic enzymes, membrane transport

Signal

Reception

Transduction

Response(s)

Amplification

Specificity of Biosignaling

• Molecular complementarity between signal and receptor– multiple non-covalent interactions similar to substrate-enzyme,

solute-transporter, and antigen-antibody interactions

• Cell-specific expression of receptors– only cells with receptors specific for the signal can respond

• Cell-specific expression of signal transduction proteins– same signal-receptor may activate or inhibit depending on other

signal transduction proteins present

• Cell-specific expression of effector proteins– differential response of liver, skeletal muscle and adipose cells

to epinephrine depends on expressed enzymes

Analysis of Receptor-Ligand Interaction

Rreceptor

RL receptor-ligand

complex

k1

k-1

Bmax

1 + Kd / [L][RL] =

Kd is [L] at ½Bmax

+ Lligand

Total [receptor], RT = [R] + [RL] = Bmax

Kd =[R][L]

[RL]=

k-1

k1

=(RT – [RL])[L]

[RL]

[RL]

[L]

[Bound]

[Free]=

Bmax – [RL]

Kd

=

Scatchard Plot Saturation Plot

slope = – 1 / Kd

Analysis of Receptor-Ligand Binding

• Saturation plot of bound receptor-ligand with increasing ligand concentration

• Scatchard plot for graphically measuring Kd and total number of receptors (Bmax)

Measurement of Insulin Binding to Receptor

• Assay of insulin binding to liver cells for measuring Kd and number of receptors per cell

• Increasing amounts of [125I]insulin added to liver cells

• Incubate at 4ºC for 1 h; separate bound and unbound [125I]insulin

• Curve A shows total bound insulin• Control assay of [125I]insulin with

100x excess unlabeled insulin for non-specific binding (curve C)

• Difference is specific binding (curve B)

Analysis of data indicates

Kd ~ 1.4 x 10-8 M

~ 33,000 receptors/cell

Sensitivity of Biosignaling

• High affinity of ligand (signal) for its receptor– Kd for receptor-ligand > [ligand] under unstimulated conditions

• Cooperativity of ligand-receptor interaction– multiple ligands to single receptor (acetylcholine)– dimerization of receptors to one ligand (cytokine)

• Receptor occupancy at maximum physiological response– relatively few receptors occupied per cell for biological activity

• Amplification of signal by enzymatic cascades– multiple levels of enzymes activating enzymes resulting in

geometric amplification of input signal

Natural Hormones and Synthetic Analogs

• Norepinephrine (prohormone)– biosynthetic precursor of

epinephrine

• Epinephrine (hormone)

• Isoproterenol (agonist)– binds to receptor and has

normal biological activity

• Propranolol (antagonist)– binds to receptor but has

no biological activity

Norepinephrine

Biological Activity and Ligand Binding

a) Biological activity of Isoproterenol (IP), Epinephrine (EP) and Norepinephrine (NEP)

b) Binding of ligands to receptor, measured by competition assay with constant amount of [3H]alprenolol (Kd ~ 3 x 10-9 M) and increasing IP (Kd ~ 2 x 10-6 M), EP (Kd ~ 5 x 10-5 M) and NEP (Kd ~ 5 x 10-4 M)

Biological Activity and Receptor Occupancy

• 50% of maximum biological activity with ~18% of receptors occupied

• >80% of maximum biological activity with 50% of receptors occupied

• Epinephrine levels of ~10-10 M can stimulate gluconeogenesis in liver cells, despite its relatively low binding affinity (Kd ~ 10-5 M)

Signal Transduction: Common Themes

Protein and metabolite carriers involved in transducing, amplifying and transmitting signal from receptor-ligand

• GTPase switch proteins (G proteins, Ras proteins)

• Second messengers (cAMP, cGMP, DAG, IP3, Ca2+)

• Cascades of Tyr and Ser/Thr kinases and phosphatases

Clustering of receptors and signal transduction proteins

• Adapter protein domains (synapses, scaffolds)

• Lipid rafts (caveolin, sphingolipid, cholesterol, PIP, GPI protein)

Interaction and regulation of signaling pathways

• Receptor-ligand specific, cell specific

• Signal integration, desensitization (receptor endocytosis)

GTPase Switch Proteins

• Structure of Gsα with

– GDP (inactive) and – GTP (active)

• 3 switch peptides close on GTP ( phosphate) when it replaces GDP

• Some require GEF (guanine nucleotide exchange factor) for activation and GAP (GTPase activating factor) for inactivation

GTPase Switch Proteins

• Activation-Inactivation cycle for Ras and Ras-like proteins

• Requires a guanine nucleotide exchange factor (GEF) and GTP for activation

• Requires a GTPase activating protein (GAP) for inactivation

• Activated G protein coupled receptors have GEF activity

• Gα subunits have intrinsic GTPase activity (time delayed)

• Some downstream effectors have GAP activity

Lipid Rafts and Signal Transduction

• microdomains on surface of plasma membrane

• segregate proteins based on attached lipid – acylated proteins in raft

– prenylated proteins not

• caveolin causes inward curvature forming caveolae

• localized in lipid rafts / caveolae:– G-protein coupled receptors

– Tyr kinase receptors (some)

• not in lipid rafts:– Ras and G subunit (prenylated)

Signaling with G-Protein Coupled Receptors

Receptors (GPCRs)• integral membrane proteins with 7 transmembrane segments• binding site for diverse ligands (hormones, odorants, tastants, light)• >907 human GPCRs (384 olfactory receptors)

G Proteins• trimeric complexes of , and subunits (20 G, 5 G, 12 G) • G is GTPase switch protein (GDP off / GTP on)• attached to membrane: G is acylated: G is prenylated

Effectors• adenylate cyclase, phospholipase C, phosphodiesterase, channels

• control levels of secondary messengers (cAMP, cGMP, DAG, IP3)

β-Adrenergic Receptor Signal Transduction

Signal transduction of epinephrine depends on the type of receptor:

β-adrenergic receptors stimulate adenylate cyclaseα1-adrenergic receptors inhibit adenylate cyclaseα2-adrenergic receptors stimulate phospholipase C

G Protein Activation Cycle

Activated receptor is the guanine nucleotide exchange factor (GEF)

Some effectors are GTPase activating proteins (GAP)

Interaction of Gsα and Adenylate Cyclase

• Structure of adenylate cyclase 2 cytoplasmic domains (blue) bound to

← forskolin (yellow) locks adenylate cyclase in active conformation

← Switch helix of Gαs docks with activated adenylate cyclase

• Structure of activated Gαs subunit with bound GTP (red)

Activation of Protein Kinase A with cAMP

Structure of PKA catalytic subunit with bound peptide substrate

R subunit domain inhibits catalytic subunit by binding to substrate site

Epinephrine Cascade

• extracellular [epinephrine] > 0.1 nM (10-10 M) is sufficient for activation

• intracellular [cAMP] = 1 μM (10-6 M)• PKA: GPK: GP ratio = 1: 10: 240

• PKA inactivates– glycogen synthase (GS)– phosphoprotein phosphatase (PP)

• at low [cAMP]: PP is activated– PP activates GS– PP inactivates GPK and GP

high cAMP: glycogenolysis

low cAMP: glycogenesis

GPK

GP

GPCRs with Adenylate Cyclase as Effector

GPCR signals with stimulatory Gsα

• Epinephrine (β-adrenergic)

• Glucagon

• Corticotropin (ACTH)

• Corticotropin-releasing hormone

• Histamine H2

GPCR signals with inhibitory Giα

• Epinephrine (α1-adrenergic)

• Prostaglandin E1 (PGE1)

• Adenosine A1

• Somatostatin

Regulation of Signaling from GPCRs

• Receptor-ligand affinity decreases with GTP exchange– dissociation of ligand terminates signal

• GTP bound to Gα is rapidly hydrolyzed– GAP activity of effector stimulates GTPase activity

• Second messenger is inactivated– cAMP phosphodiesterase hydrolyzes second messenger

• Restricted localization of signaling proteins (anchoring)– A kinase associated proteins (AKAPs) anchor PKA and PDE to

subcellular locations

• Continuous binding of ligand to receptor is required for sustained signal transduction

Desensitization of β-Adrenergic Receptors

• Continous stimulation with ligand leads to desensitization

• effector kinases modify signaling and non-signaling receptors heterologous desensitization

• GPCR kinases e.g. β-adrenergic receptor kinase (βARK) modify activated receptor inhibiting ithomologous desensitization

• arrestins e.g. β-arrestin (βarr) bind phosphorylated receptor blocking G protein signaling

• Receptor-arrestin complex may be removed from membrane by endocytosis

Desensitization modulates physiological response

Regulation of Gene Activity by GPCRs

cAMP independent pathways:• βarrestin forms scaffold for

alternative signaling pathways• Mitogen activated protein (MAP)

kinases provide signaling cascade for gene regulation

• Raf-1→ MEK1 → ERK1/2 → cell responses, e.g. cell proliferation, differentiation, and survival

cAMP-dependent pathway:• PKA translocates to nucleus and phosphorylates transcription factor

CREB (cAMP response element binding) protein• CREB bindings to CRE’s on DNA for initiating transcription

Bacterial Toxins Disrupt G Protein Signaling

Cholera toxin (Vibrio cholera) • transfers ADP-ribose from NAD+ to Gsα

• blocks GTPase activity; Gsα is always active (bound GTP)

• adenylate cyclase activation keeps [cAMP] high for days

• intestinal epithelium secretes excess Cl–, HCO3– and H2O

Pertussis toxin (Bordetella pertussis)

• transfers ADP-ribose from NAD+ to Giα

• blocks GTP exchange; Giα is always inactive (bound GDP)

• adenylate cyclase not inhibited; causes high [cAMP]

• pathology localized to respiratory epithelium (whooping cough)

Group Problem

• Why would individuals with a recessive gene for cystic fibrosis be resistant to cholera?

• How may cholera toxin and pertussis toxin be used for distinguishing which G protein is used by a receptor for signal transduction?

Fluorescent Proteins

• Structure of green fluorescent protein (GFP) from jellyfish

• Chromophore is autocatalytically formed by cyclizing and oxidizing SYG sequence

• Site directed mutagenesis of GFP produced variety of other fluorescent proteins of different wavelengths

• Combination of two FPs is basis of Fluorescence Resonance Energy Transfer (FRET) assays for protein interactions in vivo

Assay for Measuring Protein Interactions

• Fluorescence Resonance Energy Transfer (FRET) uses emission of one chromophore as excitation for a second chromophore

• If proteins interact excitation of 1st chromophore gives emission of 2nd

• Applied to signal transduction study

Selecting for Protein Interactions

Yeast 2 Hybrid System• Make construct encoding Gal4 binding domain fused to bait protein• Make cDNA library of coding regions fused to Gal4 activation domain• Transform 1st construct into yeast (select marker 1) • Transform library into transformed strain (select markers 1,2)• Select double transformants for reporter gene

GPCRs Activating Phospholipase C

• Receptors: Epinephrine (α2-adrenergic), Glutamate, Histamine H1, Acetylcholine (muscarinic M1)Platelet derived growth factor, Oxytocin, Vasopressin

• G Proteins: Gq or Go plus G (various)

• Effector: Stimulates Phospholipase C (β isoform) hydrolyzes phophatidylinositol 4,5-bisphosphate (PIP2)

• Messengers: Diacylglycerol (DAG), inositol 1,4,5-trisphosphate (IP3),

Ca2+ release• Targets: Protein kinase C (PKC) activation

Calmodulin (CaM) – regulatory subunit of enzymesCa2+/CaM-dependent protein kinases (CaM kinases)

• Response: PKC has various metabolic and cell proliferation targetsSustained activation of PKC by phorbol esters interferes with normal cell growth and division (tumorigenic)

GPCR Signaling by Phospholipase C

• Receptors use Gqα or Goα proteins

• Activated Gqα-GTP activates its effector phospholipase C (PLC)

• PLC hydrolyzes PIP2 (phosphatidyl- inositol 4,5-bisphosphate) giving 2 second messengers:– diacylglycerol (DAG) and

– inositol 1,4,5-trisphosphate (IP3)

• IP3 opens ligand-gated Ca2+ channel on ER; cytosolic [Ca2+] ↑

• Ca2+ and DAG activates protein kinase C (PKC) on membrane

• PKC phosphorylates cellular response proteins

Regulation of Cytosolic [Ca2+]

• IP3-gated channels in ER release Ca2+ into cytosol

• cytosolic [Ca2+] lowers affinity of gated channels for IP3

• causes oscillation in cytosolic [Ca2+]• cytosolic [Ca2+] measured using

fluorescent Ca2+-binding dye• Time course of cytosolic [Ca2+] with

α1-adrenergic receptor stimulation by epinephrine

• high sustained Ca2+ release may be toxic

Calmodulin (CaM)

• Ubiquitous regulatory protein for transducing effects of Ca2+

• 4 high affinity Ca2+ binding sites (Kd ~ 10-6 M)

• Regulates enzymes at helical CaM binding sites; binding involves conformation change

• CaM is member of Ca2+ binding protein superfamily including troponin

Structure of Calmodulin: • binding to 4 Ca2+

• binding to regulated enzyme (red helix)

• EF hand folding motif

Proteins Regulated by Ca2+ and Calmodulin

Signal Transduction Proteins• Adenylate cyclase (brain)• Ca2+/Calmodulin-dependent protein kinases (CaM kinases I-IV)• Calcineurin (phosphatase allowing nuclear translocation of

NFAT)• cAMP phosphodiesterase• cAMP-gated olfactory channel• cGMP-gated Na+ and Ca2+ channels (retinal rod and cone cells)

• IP3-gated Ca2+ channel

• NO synthase (paracrine signaling to vascular smooth muscle cells)

• Phosphoinositide 3-kinase• Protein kinase C (PKC)

Signal Transduction in Sensory Reception

• Vision: – GPCRs activate phosphodiesterase that hydrolyzes cGMP– Closes cGMP-gated ion channels; hyperpolarizes membrane

• Olfaction:– GPCRs activate cAMP or DAG/IP3 pathways; opens ligand-gated

Ca2+ channels and Ca2+-gated Cl– channel; depolarizes membrane

• Gustation:– GPCRs activates AC, PDE and/or PLC; depolarizes membrane– Ion-gated ion channels depolarize membrane

• Hearing:– Mechanosensory gated ion channels depolarize membrane

Light Reception by the Eye

• Photoreceptor cells– Rods: sensitive to light– Cones: less sensitive but

discriminate wavelengths 3 types: red, green, blue

• Rods and cones have same pigment (11-cis retinal) but different apoproteins (opsins) that shift of activating light

Absorption Spectra of GPCRs

Light-Induced Hyperpolarization of Rod Cells

Resting cells: high [cGMP] open Na+ channels Vm = -45 mV

Excited cells: low [cGMP] closed Na+ channels Vm = -75 mV

Interaction of Rhodopsin and G Protein

• 3D structure of rhodopsin docked with G protein (transducin)

• chromophore 11-cis retinal (blue) covalently bound to rhodopsin

• analogous location for ligands• light induced change from 11-cis

to all-trans alters conformation of rhodopsin activating G protein

• note: palmitoylation of rhodopsin, N-term myristoylation of G and C-term prenylation of G

Signal Transduction of Light

Desensitization of Rhodopsin

• Rod cells detect light over a range of 105 fold; requires adaptation

• Rhodopsin kinase phosphorylates activated rhodopsin

• Gradually reduces activation of Gtα

• Binding of arrestin-1 to phosphorylated rhodopsin turns off receptor

• Cone cells used for sight in bright light can also be desensitized

low light ATP ADP

rhodopsin kinase

high light

arrestin

very high light

*

P P P

*

arrestinP P P

Rhodopsin(dark adapted)

Activated Rhodopsin

activation of Gtα

* *

P

slightly reduced Gtα activation

greatly reduced Gtα activation

no Gtα

activation

Activated Rhodopsin

(light adapted)

Signaling by Olfactory GPCRs

• Ligands:– >1000 different odorants detected by humans

• Receptors:– ~380 G protein coupled olfactory receptors (human)– broad, overlapping specificity for odorants

• Signaling:– Golf activates adenylate cyclase → cAMP pathway

– Gq activates phospholipase C → IP3/DAG pathway

– opens ligand-gated Ca2+ channel; Ca2+ opens ion-gated Cl– channel; depolarizes membrane

Transduction of Olfactory Ligands

Gustatory Signal Transduction

• Sweet

– GPCRs with Ggust activating AC, cAMP activates PKA, PKA phosphorylate (closes) K+ channel depolarizing membrane

• Bitter– G activates cAMP-phosphodiesterase, inactivating cAMP

– G activates PLC, producing DAG and IP3, releasing Ca2+

• Umami– GPCRs activate cAMP-phosphodiesterase, inactivating cAMP

• Salty– Na+ gated Na+ channel opens, depolarizing membrane

• Sour– H+ opens gated H+ and Na+ channels, closes K+ channel,

depolarizing membrane

Transduction of Sweet Tastants

Diverse Signals – Analogous Signaling

Signal:

Receptor:

G Protein:

Effector:

Messenger:

Target:

Summary of GPCR Signaling Pathways

Signaling with Receptor Enzymes

• Ligands– Peptide or proteins; hormones, growth factors, cytokines

• Receptors– Single transmembrane α helix; often form dimers, oligomers– Intrinsic or associated protein kinase or phosphatase

• Signal transduction– Direct activation of cytosolic transcription factors– Ras-MAP kinase pathway

– IP3/DAG pathway

– PI-3 kinase pathway– Activation/inactivation of cytosolic protein tyrosine kinases

Major Classes of Enzyme Receptors

• Receptor Tyrosine Kinases– insulin, epidermal growth factor, fibroblast growth factor, growth factors

• Cytokine Receptors– erythropoietin, growth hormone, cytokines, interferons, interleukins

• TGFβ Receptors– transforming growth factor β (TGFβ) superfamily members

• Receptor Guanylyl Cyclases– atrial natriuretic factor and related peptide hormones

• Receptor Phosphotyrosine Phosphatases– pleiotrophins and related protein hormones

• T-cell Receptors– major histocompatibility complex (MHC) associated peptides

Receptor Tyrosine Kinases

• Ligands:– insulin, epidermal growth factor, fibroblast growth factor,

neurotrophins, other growth factors

• Receptors:– 2 extracellular ligand binding domains per receptor– intrinsic tyrosine kinase in cytosolic domain– cross-phosphorylation of dimer on activation

• Signal Transduction:1) Ras–MAP kinase pathway

2) IP3/DAG pathway

3) PI-3 kinase pathway

Activation of Protein Tyrosine Kinases

• Activation of a Tyr kinase by phosphorylation

Phosphatidylinositides in Signal Transduction

Ptdlns

ATP ADP

PI-4KPtdlns4P

ATP ADP

PIP-5KPtdlns(4,5)P2

ATP

ADP

PI-3KPTEN

Pi

Ptdlns(4,3,5)P3

DAG Ins(1,4,5)P3

PLC

(PIP2)

(PIP3)

[Ca2+] ↑PKC (Active)

PKB (Active)

PI-3 Kinase Pathway

(IP3)

IP3/DAG Pathway

Activation of Protein Kinase C

Activation of Protein Kinase B

Regulation of Gene Expression by Insulin

Ras–MAP kinase pathway

1) Each receptor Tyr kinase phosphorylates its partner

2) Active receptor phosphorylates IRS-1

3) Active IRS-1 binds SH2 of Grb2; Sos binds SH3 of Grb2; Sos (GEF) binds to Ras; GTP replaces GDP

4) Active RAS binds (activates) Raf-1

5) Active Raf-1 phosphorylates MEK; Active MEK phosphorylates ERK

6) Active ERK translocates to nucleus; phosphorylates transcription factors

7) Active transcription factors initiate expression of new genes

Activation of Glycogen Synthase by Insulin

PI-3 Kinase Pathway

Binding Modules of Signaling Proteins

Insulin-Induced Signaling Complex

Domains: SH2/PTB SH3 PH

Ras-MAP Kinase Pathway

PI-3 Kinase Pathway

IP3/DAG Pathway:SH2 of PLC binds receptorPLC : PIP2 → DAG + IP3; [Ca2+] ↑SH2 of PKC binds receptor;Ca2+ activates PKC

Cytokine Receptors

• Ligands:– interferons, erythropoietin, growth hormone, other cytokines,

some interleukins

• Receptors:– Conserved multi β strand fold in extracellular domain– JAK kinase associated with cytosolic domain– cross-phosphorylation of JAK kinases on activation

• Signal Transduction:1) Direct activation of cytosolic STAT transcription factors

2) Ras–MAP kinase pathway

3) IP3/DAG pathway

4) PI-3 kinase pathway

JAK – STAT Signal Transduction

JAK-STAT pathway

1) Binding of erythropoietin causes dimerization of receptors and recruitment of soluble JAK kinase to cytosolic domain

2) JAKs are phosphorylated (activated) and phophorylate receptors

3) STATs bind to receptors (SH2) and become phosphorylated

4) Phosphorylated STATs form dimers activating NLS (nuclear localization)

5) STAT dimers activate transcription of EPO specific genes

Cytokine Signal Transduction Pathways

Cytokine → Receptor → JAK →

→ STAT → Transcriptional Activation

→ Grb2 → Ras → MAPK →

Transcriptional Activation / Repression

→ PLC → IP3 → [Ca2+]↑ →

Modification of Cellular Proteins→ PI-3 Kinase → PKB →

• Same receptor ligand may have different signaling in different cells• Different signaling paths in same cell may have opposing effects

Termination of Tyrosine Kinase Signaling

• Endocytosis of receptor-ligand complexes• Phosphatases:

– of receptor kinases: SHP-1, SHP-2– of MAP kinases and STATs

– of PIP3: PTEN turns off PI-3K pathway (PKB)

• SOCS (CIS) proteins: binds to phosphotyrosines on receptors– SOCS-1 (erythropoietin), SOCS-2 (growth hormone)– inhibits JAK kinases and binding of signaling proteins (e.g. Grb2)– promotes polyubiquitination and receptor degradation

• Ras GAP activates Ras GTPase

Group Study Problem

• Erythropoietin (EPO) is produced by the kidney in response to low pO2; it stimulates erythrocyte production in bone marrow

• EPO is used for treating anemia particularly following chemotherapy, but has been abused for enhancing athletic performance

• Blood doping with EPO increasing hematocrit levels from normal levels of ~46% to >60%, greatly improving delivery of O2 to muscles

• A Finnish cross-country skier, winning several medals in the 1960s, had a hematocrit >60%, but below-normal levels of EPO

• Genetic analysis of the skier and his family revealed an inherited defect in the gene encoding the EPO receptor

• The encoded receptor could transduce the EPO signal normally but could not bind to the SHP-1 protein

• How could this defect result in higher than normal erythrocyte counts but lower than normal erythropoietin levels?

Receptor Guanylyl Cyclases

• cGMP synthesized by guanylyl cyclase on receptors • cGMP is a second messenger with various cell-specific functions• cGMP mediates effects of atrial natriuretic factor (ANF) in kidney

– ANF is produced by distended right artium (high blood volume)– Binds to membrane receptors on collecting duct cells of kidney– cGMP synthesized by ANF receptor increases excretion of Na+

and water (reducing blood volume) • cGMP mediates vasodialation of vascular smooth muscle by NO

– Activated NO receptor (in cytosol) produces cGMP activating a cGMP-dependent protein kinase (protein kinase G, PKG)

– PKG has catalytic and regulatory domains on same protein and is regulated by mechanism analogous to PKA

Cyclins and Cyclin-Dependent Kinases

Phases of Eukaryotic Cell Cycle • Cyclin-dependent kinase (CDK) activities control entry into phases of cycle

• G1 → S: Cyclin E-CDK2• S → G2: Cyclin A-CDK2• G2 → M: Cyclin B-CDK1

Activation of Cyclin-Dependent Kinases

2-Step activation of CDKs by cyclin binding and phosphorylation

Cyclin Binding Phosphorylation

T loop phosphorylation (orange) stabilized by Arg (red) increasing kinase activity

Binding of cyclin (blue) changes folding of T loop and N-term helix (green) activating kinase (~104 fold)

Inactive CDK2 without cyclin:

T loop (red) blocks substrate binding site near to ATP (light blue)

Mechanism for Regulating CDK Activity

• Phosphorylation of CDKs– phosphorylating Tyr near ATP binding site inhibits– phosphorylating T loop blocking substrate binding site activates

• Degradation of cyclins– DBRP (destruction box recognition protein) directs ubiquitination– DBRP is activated (phosphorylated) by active CDK

• Synthesis of CDKs and cyclins– e.g. TF E2F required for cyclin D, cyclin E, CDK2, CDK4

• Inhibitors of CDKs– P21 binds/inactivates cyclin-CDKs at DNA damage checkpoints

– Cyclin D-CDK4/6 (G1), Cyclin E/A-CDK2 (S), Cyclin A/B-CDK1 (M)

Control by Phosphorylation and Degradation

Control by Transcription and Inhibitors

ATM ATR