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Biochemistry 503 November, 2008Protein Kinases

andProtein Phosphatases

David L. Brautigan db8g@virginia.eduCenter for Cell SignalingDepartment of Microbiology

Today: Protein Ser/Thr Kinases

I. Selected History of Cell Signaling (inc. UVA)II. Simple Basis for Enzyme RegulationIII. Structure and Mechanism of

Protein Ser/Thr Kinases

Assigned reading for this class session:Chapter 32 in Biochemistry by Garrett & Grisham

or Chapter 20 in Molecular Cell Biology by Lodish et alor Chapter 15 in Molecular Biology of the Cell by Alberts et al.

PLUSTaylor SS, Yang J, Wu J, Haste NM, Radzio-Andzelm, G.PKA: a portrait of protein kinase dynamics.Biochim Biophys Acta. 2004 1697:259-69. PMID: 15023366

I. History Molecular Mechanisms of Hormone Action.1. (1930-40’s) Regulation of blood glucose by adrenaline in liver

slices and tissue homogenates.Glycogen is storage polymer of glucoseGlycogenolysis response to hormones requires membranes.Forms glucose esters: glc 1-phosphate and glc 6-phosphate (Cori & Cori)

Glycogen(n) + Pi Glycogen(n-1) + Glc 1-P

Gerty & Carl CoriWashington Univ. St. Louis, MO(Nobel Prize)

Gerty Cori1st American womanto win Nobel Prize in Science

2. (1940-50’s) Phosphorylase catalyzes glycogen phosphorolysis. Phosphorylase

Glycogen(n) + Pi Glycogen(n-1) + Glc 1-P FIRST example of enzyme with

multimeric subunit control (dimer-tetramer)allosteric regulation (G6P, AMP)cofactor action (pyridoxal phosphate action)non-reversible biosynthetic pathway

(1955) protein phosphorylation - ATP used to transfer PO3 to Ser14phos b kinase

Phosphorylase b (inactive) Phosphorylase a (active)

PR enzyme

Edwin (Ed) Krebs (left) andEdmond (Eddy) Fischer (right)

Nobel Prize 1992

Filter Paper Experiment leads to Nobel Prize

3. (mid 1950’s) Cellular “second messenger” (cyclic AMP = cAMP) A heat-stable, acid-stable low MW factor that mimics hormone addition

to homogenates, no membranes required…….hormone “first messenger” actionis to produce cAMP in membrane fraction

(Earle Sutherland, Nobel Prize)

4. (1960-1970’s) Second Messenger Hypothesis

Hormone --> receptor -->transducer -->catalytic action--->second messengerbeta-agonist-> GPCR --> G protein ---> adenyl cyclase ---> cAMPhormone ----> GPCR ---> G protein ---> phospholipase ---> DAG + IP3--> Ca2+

(Martin Rodbell, Nobel Prize) (Michael Berridge- sorry! No Nobel Prize)

Hormone cAMPBlack

Box

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7. (1968) Purification and properties of the cAMP-dependentprotein kinase (PKA) ….the target for cAMP Ed Krebs and Donal Walsh

PKA activates phosphorylase kinase, the activator of phosphorylaseThis 2 step kinase “cascade” connects cAMP to glycogenolysis

cAMP---> PKA PhosKinase phosphorylase---> glycogen--> Glc-P

10-9 M hormone to 10-3 M glucose - a million-fold amplifier

PKA also phosphorylates other metabolic enzymes (studied in 1970’s-1980’s)Pyruvate KinaseAcCoA CarboxylaseGlycogen SynthaseHMG CoA reductase

cAMP via PKA produces a general catabolic response-- metabolic regulation

R2C2 + 4 cAMP = R2(cAMP)4 + 2C

University of Virginia - School of Medicine leadership in signal transduction - cell signaling

Mechanisms of Insulin Action - Diabetes glycogen synthesis - glycogen synthaseregulation by protein phosphorylation

5. (1970’s) GTPases as Hormone Signal Transducers in Membranes cyclase activation required both GTP and ATP detergent-extracted protein active in GTP form

heterotrimeric αβγ forms dissociated to α + βγ by hormone

6. (1970’s) Nitric Oxide (NO) as activator of G-Cyclase (cGMP) Formation of cGMP from GTP increased [cGMP] relaxes smooth muscle (improves blood flow).

basis for treating pulmonary hypertension in infants how Nitroglycerin works to assist heart failure

(also basis for Viagra/Cialis/Levitra for erectile function)

Al Gilman (Nobel Prize with Marty Rodbell, at NIH)

Ferid Murad (Nobel Prize)

Joe LarnerChair of PharmacologyUVA-SOM

Son, James Larner now Chair of Radiation Oncology UVA

University of Virginia leadership in signal transduction8. (1980’s) The MAPK (ERK) pathway

(Tom Sturgill)biochemical purification ofkinase activated by insulingave a 42 kDa Ser-Thrkinase reactive with MAP-2

(Mike Weber) v-src oncogenephosphorylates Tyr ina 42 kDa protein• MAPK is a kinase dual

phosphorylated on Thr-x-Tyr• activation of MAPK by MEK, adual-specificity kinase• 3 major pathways: MAPK, JNKand p38MAPK

University of Virginia leadership in signal transduction

9. (1990’s to present) Viral oncogene src = Protein Tyr Kinase

J. Thomas Parsons Sarah (Sally) Parsons

Monoclonal antibodies to individual tyrosine-phosphorylatedprotein substrates of oncogene-encoded tyrosine kinases.Proc Natl Acad Sci U S A. 1990 87: 3328–3332.PMCID: PMC53893 Potentiation of epidermal growth factor receptor-mediated

oncogenesis by c-Src: implications for the etiology of multiplehuman cancers.Proc Natl Acad Sci U S A. 1995 92: 6981–6985.PMCID: PMC41455

Susan Taylor(UCSD)US National Academyof Sciences

First 3D structure of anyProtein Kinase (1991)

PKA catalyticsubunit

Human Genome~ 500 Protein Kinases

II. Enzyme RegulationA. Catalysis - ancient function for any 3D structure• binding of substrate molecules to specific surface site• binding of vitamins as active site co-factors, expands chemistry• chemical reactions: breaking and making covalent bonds• biosynthesis coupled to ATP hydrolysis (>2 substrates)

substrate

productenzyme

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B. Suppression of catalysis - basis for regulation

1. Occlusion of active site - steric exclusion

a. intrasteric - same polypeptide folding on itself,e.g. PKC, CaMK

b. multimeric - separate subunits regulatory-inhibitory,e.g. PKA

2. Distortion of active site. Conformationalcontrol reduces substrate binding orimpairs catalysis

X

C. Relief of Suppression - equivalent to activation

1. Allosteric control (effect of added X factor)a. ligand-induced, eg. cAMP, diacylglycerolb. protein-induced, eg. CaM, cyclins

2. Covalent controla. reversible - phosphorylation, acetylation, methylation,

nitrosylation, oxidationb. irreversible - proteolysis, zymogen activation, caspases

inactive

substrate

product

D. Allosterism and Cooperativity in Enzyme Control The molecular theme from hemoglobin [α2β2]Regulatory Proteins have 2 pairs (2x2) of interacting binding sites.protein Hb = PKA-R2 = CaMligand oxygen cAMP Ca2+

Hemeglobin binds O2 to each α and β chain Check out O2 saturation to Hb in textbook

Hb + 4 O2 ---> Hb(4O2)

cAMP-dependent protein kinase (PKA) R2C2 + 4 cAMP ---> R2[cAMP]4 + 2CDimers of R; each R has 2 sites for cAMP

Calmodulin has two pairs of Ca2+ binding sites in one protein chain, a 2x2 arrangement. CaM + 4 Ca2+ --> CaM[Ca2+]4

Kinases

[2nd messenger] for activation

resp

onse

non-cooperative

cooperative

cooperativenon-cooperative

III. Protein KinasesA. Enzymes that use MgATP as substrate

1. ATPases - use water to hydrolyze ATP to ADP, couple to worka. Molecular motors

myosin + actindyenins and kinesins

b. Pumps that transport ions with ATPase activityNa+/K+

Ca2+ (SERCA)H+ proton pumps

2. Kinases - phosphoryl group transfer from ATP to second substratea. Metabolite kinases

hexokinase : Glc + ATP--> Glc 6-P + ADPPFK F6-P + ATP --> F 1,6-diP + ADP

b. Protein Kinases - a Superfamily of Enzymes1. Common Features

extraordinary conservation among eucaryotesyeast -worms-flies-vertebrates-mammals

reflects common protein 3D structure for catalytic activity

2. Different features separated by functional properties and sequence variations landmarks of different substrate specificity distinctive modes of regulation

8 major groups see Hunter Science 298: 1912 (2002)a. Ser-Thr kinasesb. Tyr kinasesc. His (Asp) kinases in 2-component signalingd. other : Lys, Asp, Arg, etc.

Protein KinasesB. Catalytic site mechanism

1. Residues essential for kinase function• Lys (K) at active site for binding + to beta phosphate of ATP• Asp (D) as general base to remove H from Ser or Thr• Glu (E) in “C” helix to close active site on ATP

O OO-P-O-P-O O O O P -- O

OH-O- Ser

O

AdMg2+

HO OH

K72

D166

E91C

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C. Kinetics of PhosphoTransferase Reaction

MgATP

E +

Ser-OH

MgADP

Ser-OPO3

E-MgATP-SerOH

E-MgADP-Ser-OPO3

KM 10 uM

200 sec-1

KM 10-50 uM

500 sec-1

100 sec-1

slow E

enzyme-substrates complex

enzyme-products complex

D. 3D Structure of PKA Catalytic subunitThe prototype for all protein kinases

N terminal lobe (red) mostly beta sheets binds MgATP

C terminal lobe (blue) mostly alpha helix binds peptide substrate

C helix

Phosphorylation of two sites…activation loop

Protein Kinase ACatalytic Subunit

with Mg-ATP (ball & stick)

and PKI (peptide inhibitor)(yellow) same as substrate

E. Substrate-induced kinase clamp…. conformational “trapping” of substrate

MgATP at the active sitebeta-sheet N terminal domain forms cleftalpha-helix C terminal domain binds peptide and has P -loopN and C clamp together, like clamshell

C helix moves to close site; prevents hydrolysisshift seen by H-D exchange +/- ADP binding (MALDI-TOF) J. Biol. Chem. 276:14204 (2001)

E (Glu) residue in C helix essential for closed conformationmutate E to produce “dead” kinase, catalytically inactive

E. Substrate-induced kinase clamp…. The C helix produces open and closed conformations

(see assigned reading)

Front view Side view

Phosphorylation sites in “activation loop”

E. Substrate-induced kinase clamp….The C helix and Kinase Activity…

proximity of essential K and E residues

C helix

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F. Specificity of cAMP-dependent Protein KinaseSequence specificity studied by synthetic peptide substrates andanalysis of phospho-sites in proteins

PKA gives RRxS as motif

Other kinases also display preferences based on primary sequencePhosphoSite analysis, e.g. Pro-directed kinases, CDKs, MAPKs, GSKs

This sequence specificity also basis for regulation, when the kinase itself has a sequence motif that resembles substrate“pseudosubstrate” region produces intra-steric regulation of activity

G. Suppression and Relief in Kinase Regulation

1. Intramolecular pseudosubstrate with ligand relief PKA cAMP PKC diacylglycerol CaMK CaM-Ca2+ PKG cGMP

2. Conformational controlconformation at ATP binding site is criticalgeneral requirement for phosphorylation in P-loopcyclin binding - conformational activation of CDKs by

changes in N terminal lobe

Binding of R to Cinvolves a substrate-like association of Rsubunit at the activesite, blocking access ofsubstrates.

H. Targeting of cAMP-dependent Protein Kinase

Catalytic + Regulatory + Targeting 2 C + R2 = R2C2

+ AKAP (A kinase anchoring protein)

C2 R2--AKAP------> Sub-cell structures

Binding of R2 (blue/grey) to AKAP involves an amphipathic helix (red)

Multiple AKAPs Distribute PKA to Multiple Intracellular Sites(and also scaffold together PKA, other kinases and protein phosphatases)

I. Functional Types of Protein Ser-Thr Kinasessub-types based on protein sequences and activation mechanisms

> 400 enzymes in human genome

1. AGC group PKA, PKG, PKC, PKB(Akt)2. CaMK group I, II, IV, phos kinase3. MAPK group MAPK, JNK, p384. CDK group CDK 1, 2, 4, 6 etc.5. ROCK and Rho group PAK (rac), aPKC (cdc42), (Rho)6. PI3K-ATM ATR, TOR, DNA-PK

Protein Kinases in the Human Genome

Cell Signaling Technologies<www.cellsignal.com>

Protein Tyr Kinases

Protein Ser/Thr Kinases

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