Protein Kinases, the Most Important Biochemical Regulatory System in

Animal Cells• Big Picture

• Definition, History, and Gene Number

• Classifications– Serine/Threonine Kinases– Tyrosine Kinases

• MAP protein kinase networks and pathways

Sutherland Second Messenger Hypothesis

Protein Kinases in Animal Cells

• Cell division• Apoptosis

The first messengerinteracts with a receptorand a second messenger is formed

History and Importance I

• About 518 genes in humans encode protein kinases; there are an estimated 30,000 genes in humans, so that about 1.7% of the human genome encodes protein kinases

• Protein kinases are the fourth largest gene family in humans– C2H2 zinc finger proteins (3%)– G-protein coupled receptors (2.8%)– Major histocompatibility (MHC) complex

protein family (2.8%)

How Many Protein Kinases Are There? (518)

• The kinome refers to all protein kinases in the genome• There are 478 conventional eukaryotic protein kinases (ePKs)

plus 106 pseudogenes– 388 Protein-serine/threonine kinases– 90 Protein-tyrosine kinases

• 58 receptor PTKs• 32 Non-receptor PTKs

• There are 40 atypical protein kinases (e.g. EF2K/alpha kinases)• 478 + 40 = 518• The exact numbers aren’t important; understand the

classification

General Classes I

• ACG Group• Protein kinase A; cyclic AMP-dependent

protein kinase• Protein kinase C• Protein kinase G• Basic amino acid-directed enzymes that

phosphorylate serine/threonine (you don’t have to memorize any sequences)

General Classes II

• CaMK• Calcium-calmodulin-dependent protein kinases

– I– II – III – IV

• Type II is a broad specificity kinase• The others are dedicated kinases with a limited

substrate specificity

General Classes III

• CMGC• Cyclin-dependent protein kinases

– These are important regulators of the cell cycle

• MAP (Mitogen activated protein/microtubule associated protein) kinases– Many of these promote cell division

• GSK3 (glycogen synthase kinase-3)• Clk (Cyclin-dependent like kinase)

General Classes IV

• PTK (Protein-tyrosine kinases)– Receptor, e.g., epidermal growth factor

receptor, insulin receptor– Non-receptor, e.g., Src, Abl protein kinases

• Specifically phosphorylate protein-tyrosine (note they are not tyrosine kinases but protein-tyrosine kinases)

Protein Kinase Classifications• Protein-Serine/threonine• Protein-Tyrosine

– Receptor: ligand binding domain and catalytic site on the same polypeptide

– Non-receptor: catalytic domain separate from the receptor

• Dual Specificity (both serine/threonine and tyrosine) also occur

• Broad Specificity: have several substrates, e.g., PKA• Narrow Specificity: have one or a few substrates, e.g.,

pyruvate dehydrogenase kinase with one substrate• Classified by activator: PKA, PKG, PKC

Second Messengers (Fig. 19-4)

Protein Kinase and P’ase Rxns (Fig. 4-8)

Reactions and Types of Protein Kinases

• Be able to recognize the three amino acids with an –OH in their R-group– Serine– Threonine– Tyrosine

Serine/Threonine Protein Kinases

Activation of Protein Kinases

• Know PKA activation mechanism– It is the only one where there is

a dissociation of regulatory subunits from catalytic subunits

– This was the first activation mechanism to be described, but it turns out to be atypical or unique

• PKG, allosteric• PKC, allosteric

Hormones and cAMP

Many first messengers lead to changes in [cAMP]

Protein Kinase A Structure

• Bilobed– N-lobe (upper)

mostly beta sheet– C-lobe (lower)

mostly alpha helix

• Active site between the two lobes

• ATP is bound in the active site

PKA Domain Structure

PKA Substrate Specificity

Basic-Basic-Xxx-Ser-Hydrophobic is preferred

Selected Protein Kinase A Substrates• Phosphorylase kinase alpha and beta subunits• Pyruvate kinase (Liver type)• 6-Phosphofructo-2-kinase/phosphatase• Hormone sensitive lipase• Protein phosphatase inhibitor 1• CREB (a transcription factor)• Aromatic amino acid hydroxylases

– Tyrosine hydroxylase– Tryptophan hydroxylase– Phenylalanine hydroxylase– What else is special about these three enzymes?

• Raf• Grandmother

Earl W. Sutherland, Jr (cAMP)Edwin Krebs (PKA) Albert G. Gilman (G-protein)Edmund Fischer (PKA) Martin Rodbell G-protein)

Enzyme Cascades and Phosphorylase and Synthase

• Hormonal regulation • Hormones (glucagon, epinephrine) activate adenylyl cyclase

– Glucagon, liver– Epinephrine in muscle

• cAMP activates kinases and phosphatases that control the phosphorylation of phosphorylase and glycogen synthase

• GTP-binding proteins (G proteins) mediate the communication between hormone receptor and adenylyl cyclase

• Learn the regulation of the PKA-phosphorylase cascade!!!

cAMP Metabolism

Regulation of Glycogen Metabolism

PKA phosphorylase kinase phosphorylaseA kinase acting on a kinase that phosphorylates a protein is a cascadeSteps 3 and 4 make up the first cascade to be described

Protein Kinase G

• Activated by cGMP• Second second messenger protein kinase (after

cAMP)• Little known about physiological protein

substrates despite extensive investigation• Two types of guanylyl cyclase

– Second messenger generated by atrial naturetic factor as an integral membrane guanylyl cyclase

– Second messenger generated by NO action on the soluble guanylyl cyclase reaction

Action of cGMP

Review Fig 19-23 forNO biosynthesis

Cyclic GMP Metabolism (Fig. 19-24)

• Integral membrane guanylyl cyclase (ANF {atrionaturetic factor} receptor)

• Soluble: Activated by NO

cGMP Metabolism and Action

Regulation of Protein Kinases G(Equation 19.1)

Selected Protein Kinase G Substrates

• G substrate (cerebellum)

• Vasodilator-stimulated phosphoprotein

• Most substrates and their functions are unknown

Calcium-Dependent Protein Kinases• Protein Kinase C

– Requires calcium, diacylglycerol, and phospholipid for activity

– Diacylglycerol generated by the action of phospholipase C

– Activated by phorbol esters (tumor promoters)

– Many isozymes

• Calcium-calmodulin-Dependent Protein Kinases– CAM Kinases I, II, III, IV

– Several other protein kinases activated by calmodulin including myosin-light chain kinase, phosphorylase kinase, some isoforms of adenylyl cyclase and some isoforms of phosphodiesterase

Calcium-Dependent PKs

Hormones and Phospholipase C• Heterotrimeric Gq activates PLC

No need to memorize any of these

Hydrolysis of PIP2

• Two second messengers are generated– DAG activates PKC

– IP3 leads to a rise in cytosolic Ca2+

– This activates PKC and CaM Kinases

• Fig. 12-6

Protein Kinase C Family

• PKC– C refers to calcium– DAG and phospholipid

were also described as necessary for the activation of this enzyme

– There are many isozymes that are products of different genes

• It is paradoxical to have PKCs that are independent of Ca2+ and DAG

Selected Protein Kinase C Substrates• Glycogen synthase (at least two sites,

inactivates)• PDGF receptor• EGF receptor• Insulin receptor• Transferrin receptor• Ribosomal protein S6• Raf• No need to memorize any of these

Calcium-calmodulin Dependent Protein Kinases

• CAM Kinase I: synapsin I and II• CAM Kinase II

– CAM Kinase II (autophosphorylation)– Tyrosine hydroxylase (rate-limiting for catecholamine

biosynthesis)– CREB transcription factor– Many others

• CAM Kinase III: Elongation factor II of protein synthesis (This is a dedicated protein kinase)

• CAM Kinase IV– Found in the nucleus– Also phosphorylates the CREB transcription factor– Many others

Receptor Protein-Ser/Thr LigandsTransforming Growth Factor-β Ligands

• This family has diverse functions (that you don’t need to remember)– BMP2 subfamily: BMP2 and BMP4, chondrogenesis and other

developmental functions– BMP5 subfamily: BMP5,6,7,and 8, development of nearly all organs– BMP3/osteogenin subfamily: bone formation– Activin subfamily: erythroid cell differentiation– TGF-β1, 2, and 3: control of proliferation and differentiation;

production of the extracellular matrix

• There are about 15 BMPs– They are synthesized as integral membrane proteins and the

BMP is cleaved extracellularly in a regulated fashion– Proteins contain about 450 aa; BMPs are about 110 residues– BMP-2 and BMP-4 are expressed by human adult pulp tissue– BMP 1 is a C-terminal procollagen protease

Smads• Transforming growth factor beta ligands including

the BMPs activate receptor protein Ser/Thr kinases– Smad transcription factors are phosphorylated and

activated– Smads enter the nucleus to bring about a response

• These human proteins are homologous to Drosophila proteins called Sma or Mad, thus smad

• Smads play a role in normal cell growth, cell division, and apoptosis (programmed cell death)

TGF-β: A Model for the Smad Signaling Pathway

Protein-Tyrosine Kinases• Receptor

– Insulin

– Epidermal Growth Factor

– Platelet Derived Growth Factor

• Non-receptor– Src protein kinase

– Abl and Bcr-Abl

– Jak (Janus kinase [two catalytic regions]) or whimsically, just another kinase

Human Protein-Tyrosine Kinases from the Human Genome Project

• The protein-tyrosine kinases are a large multigene family with particular relevance to many human diseases, including cance

• A search of the human genome for tyrosine kinase coding elements identified several novel genes and enabled the creation of a nonredundant catalog of tyrosine kinase genes

• Ninety unique kinase genes can be identified in the human genome, along with five pseudogenes– Of the 90 tyrosine kinases, 58 are receptor type, distributed into 20

subfamilies

– The 32 nonreceptor tyrosine kinases can be placed in 10 subfamilies

Epidermal Growth Factor Receptor Family

• In the 1950s-60s Stanley Cohen discovered a factor present in crude submaxillary preparations that induced precocious tooth development in newborn mice that he called epidermal growth factor

• He purified EGF, determined its sequence, studied its binding to the EGF receptor, showed that a single molecule contained EGF binding and protein kinase activity

• He demonstrated that the EGF receptor was a protein-tyrosine kinase, the first to be described (1980)

• He also showed that EGF and receptor are taken up by cells and are degraded in lysosomes (many receptors undergo this fate)

EGF Growth Factor and Receptor Family

• Null mutants of any family member are embryonic lethal

• Important in development

• Implicated in many cancers

ErbB and Malignancies

• Head and neck squamous cell carcinomas (>90% associated with ErbB overexpression)– 27,000 new cases in the US per year

• Bladder• Breast• Kidney• Non-small cell lung• Prostate cancers• Many other solid tumors

CA of the Tongue

• Early squamous cell carcinoma of the tongue• Malignant neoplasms of the oral cavity account for 3-5% of all malignancies

– 50% involve the tongue (lateral border and ventral surface most common)– Then floor of the mouth > gingiva > alveolar mucosa > buccal mucosa > palate

• Squamous cell carcinomas account for >90% of all malignancies of the oral cavity• Men/woman = 2/1• Usually more than 40 years of age• Under diagnosed; more than 50% have metastasized at the time of Dx

Receptor Activation

• Ligand binds, dimers form, transphosphorylation occurs, and the receptor is activated– Homodimers: ErbB1/ErbB1, etc.– Heterodimers: ERbB1/ErbB2 (common in

breast cancer)– One of the dimers phosphorylates the other, and

the other dimer phosphorylates the one

How Does the Growth Factor Activate the Receptor?

Monoclonal Abs in the Rx of Cancer• Mabs are directed toward the ectodomain of the

ErbB2/HER2 receptor• Herceptin

– 20-30% of all human breast cancers overexpress ErbB2, or HER2 (Human Epidermal growth factor Receptor)

– These tumors can be treated with Herceptin• It targets domain IV of ErbB2

• Erbitux– Treatment of colorectal cancer that has spread– In combination with irenotecan (a DNA topoisomerase

I inhibitor)– From ImClone (The Martha Stewart case)– Approved by US FDA in February 2004

Structure of the EGF Receptor Protein-Tyrosine Kinase Domain

• Open activation loop is active (blue or green)

• Compact activation loop is inactive (magenta)

• This is an important regulatory concept

• Blue: EGF unphosphorylated

• Green: IRK phosphorylated• Magenta: IRK

unphosphorylated

ErbB1 Protein-Tyrosine Kinase

Inhibitors• Irressa is approved for the

Rx of non-small cell lung cancers

• Tarceva is near approval• ATP-competitive

inhibitors• Aromatic ring systems of

OSI make a 42 degree angle when bound to ErbB1 kinase domain

Binding of Tarceva to ErbB1 Kinase Domain

The Philadelphia Chromosome

• It results from the reciprocal translocation involving chromosomes 9 and 22

• This fuses the Abl gene from chromosome 9 to the Bcr gene on chromosome 22

– This results in the transcription of an mRNA corresponding to a Bcr-Abl oncoprotein with protein-tyrosine kinase activity

– The oncoprotein produces chronic myelogenous leukemia

• Ph1 is usually the paternally derived chromosome 9 that is translocated to the maternal chromosome 22; it is a shortened chromosome 22

Bcr-Abl Protein Kinase

• The Philadelphia chromosome occurs in the granulocytes in patients with chronic myelogenous leukemia (CML)

• The resulting Bcr-Abl protein-tyrosine kinase is constituitively activated (it is active all of the time)

• Gleevec is a specific inhibitor of the Bcr-Abl protein-tyrosine kinase, and Gleevec is used therapeutically for chronic myelogenous leukemia

• This malignancy is unusual because it results from a single genetic alteration; most cancers result from multiple somatic genetic alterations

Translocation II

Gleevec

• This drug is useful in the treatment of chronic myelogenous leukemia

• It is an inhibitor of the Bcr-Abl protein-tyrosine kinase

• It is competitive with respect to the ATP substrate

• Many oncogenes are protein kinases, and there is a major effort underway to develop therapeutic agents that inhibit these enzymes

• Protein kinase C, src, and the EGF receptor are also drug targets for cancer therapy

Lineweaver-Burk Inhibition Plots

Fig. 4-6

The Activation Loop

• A, blue loop in compact, inactive conformation

• B, red loop in open, active conformation

Regulation of Normal Abl Kinase• The latch, Sh2, and Sh3

domains lock Abl into an inactive conformation

• Sh2, ordinarily binds phosphotyrosine

• Sh3, binds to proline residues

• The fusion oncoprotein lacks the normal N-terminus and the latch; the protein assumes an active conformation

Leukemia• The oral changes that occur in leukemia are related to local

leukemic infiltrations, thrombocytopenia, neutropenia, and anemia

• Left: leukemic gingivitis due to infiltration of leukemic cells, also gingival hemorrhage

• Right: pale (anemia) with petechial hemorrhages (thromocytopenia)

Chronic Myelogenous

Leukemia

• This shows marked gingival enlargement; it is more common in lymphocytic than myelocytic leukemias

• Chronic leukemias– Middle aged persons; men > women– Onset and course insidious and it is often diagnosed

accidentally during a routine blood check

MAP Kinase Kinase Kinase

MAP Kinase NomenclatureMitogen Activated Protein Kinase• Kinase: MAPK is ERK

• Kinase kinase: MAPKK is MEK

• Kinase kinase kinase: MAPKKK is Raf

Initiation of a Signal

• Ligand binding produces receptor dimerization, trans phosphorylation, and the resulting p-Tyr attracts proteins via SH2 domains

• Proteins that (may) dock with P-Tyr via SH2 domains: PLC, PI3 kinase, Shc, Grb-2, and many others– Not all RPTKs associate with all SH2-containing

proteins

• There may not be phosphorylation of other substrates by the receptor protein-tyrosine kinase

Src, A Non-Receptor Protein Tyrosine Kinase• v-Src was discovered as an oncogene of Rous sarcoma virus, a

chicken virus, and this was the first oncogene to be described as such• c-Src, the normal homolog, is activated by PDGF and CSF receptors,

which are in turn activated by their ligands and protein-phosphorylated

• Src may be activated by transmembrane receptors that lack protein-tyrosine kinase activity

• c-Src is also phosphorylated and activated during mitosis• Myristoylation of the N-terminus is required for attachment to the

plasma membrane and for activity (Src lacks a signal peptide and is found initially in the cytosol)

• The physiological substrates for c-Src are unknown despite exhaustive experimentation

• High activity in brain, a non-dividing tissue• The protein contains SH2 domains that bind to protein tyrosine

phosphates and SH3 domains that bind to proline-containing regions as we saw in Abl

Src Regulation

Protein Phosphatases (P’ases) Fig. 4-8

• These catalyze the hydrolytic removal of phosphate from proteins; these are unidirectional reactions

How Many Protein Phosphatases Are There?

• Studies from the human genome indicate that there are the following number of P’ases– 32 serine/threonine phosphatases

– 42 protein-tyrosine phosphatases

– 46 dual specificity phosphatases

• It is surprising that there are so many protein-tyrosine and dual specificity P’ases

Protein-Serine/threonine Phosphatases

Catalytic subunit Regulatory Elements

Regulated Functions

PPP FamilyPP1 >15 that target and regulate

the catalytic subunitGlycogen metabolism, muscle contraction, cell cycle, mRNA splicing

PP2A B subunits target and regulate core enzyme

MAP kinase pathway, metabolism, cell cycle

PP2B Ca/CAM activates T-lymphocyte activation, brain NMDA receptor signaling

PPM Family

PP2C Integral N or C-terminal peptides

Antagonism of stress activated kinases

PPP Family of P’ases• Contain Zn2+ and Fe2+ in the active site• M subunits target the 38-kDa catalytic subunit of PP1 to myosin• G subunits target PP1 to glycogen• PP2B, or calcineurin

– Regulated by Ca2+

– It couples Ca2+ to protein dephosphorylation– Two subunits

• A forms the P’ase active site with Zn2+ and Fe2+ • B subunit binds Ca2+ • Ca/CAM is the true activator

• PPM– Unrelated to PPP by sequence, but both use Zn2+ and Fe2+ – Evolutionary convergence

Protein-Tyrosine PhosphatasesCatalytic Subunit Regulatory

ElementsRegulated Functions

PTP FamilyCytosolic (PTP1B, SHP1, SHP2) SH2 and other domains

target to substratesVarious

Transmembrane PTPs (cd45, RPTPμ, RPTPα

Homodimerization inhibits Lymphocyte activation

Dual SpecificityCdc25 Family Polo kinase, Chk1 kinase,

phosphatasesCell cycle

Low Molecular weight (Acid phosphatases)

Located in lysosomes ?

Protein-Tyrosine P’ase• All four families bind phosphate to a sulfur in a sequence

Cys-x-x-x-x-x-Arg• Forms a covalent P-S bond• Evolutionary convergence• PTP family

– 230 residue catalytic domain

– Cytoplasmic and transmembrane members

– CD45• 10% of the plasma membrane protein of white blood cells

• Required for antigens to activate B and T cells

• May activate one or more of the Src-family tyrosine kinases associated with the T-cell receptor by dephosphorylating inhibitory phosphotyrosine residues

Protein-Tyrosine P’ase• Dual specificity

– Inactivate the MAP kinases– Substrate binding site is shallow and can attack pY, pT,

and pS

• Cdc25 Subfamily– Remove inhibitory pY and pT from CDK1 and CDK2– This dephosphorylation promotes cell cycle progression– These dephosphorylations thus have a positive effect

• Cooperation between kinases and P’ases– PP2A is bound to Ca/CAM kinase IV– MPK-3 (MAP kinase P’ase 3) is bound to ERK 2, one of

the MAP kinases

Cross-Talk between Signal Pathways

Cyclic Nucleotide Metabolism• First and second messengers• G-proteins• Adenylyl cyclases

– 9 forms of membrane bound enzyme

– 1 soluble form

• Guanylyl cyclases– 3 major forms

• ANF (Atrial naturetic factor) receptor in membrane

• Soluble form activated by NO

• Cyclic nucleotide phosphodiesterases– 11 forms with varying substrate specificity and

regulatory properties

Regulation of Adenylyl Cyclase

Functions of the Isoforms

• ACI is important in behavior and memory as shown in knock-out mice

• ACVIII knock-outs do not exhibit increased anxiety in response to stress

• ACV is important in cardiac function

• Much more remains to be done

GC and AC• Adenylyl cyclases and guanylyl cyclases show high specificity for

their respective substrates

• This is essential for the coexistence and fidelity of signaling pathways, since virtually all cells contain both enzymes.

• Guanylyl cyclases exist as both soluble and membrane-bound species. Both types of the enzyme contain cytoplasmic domains similar to those of the adenylyl cyclases

• Membrane-bound guanylyl cyclases are homodimers, whereas the soluble enzymes contain and alpha and beta subunits, both of which are required for catalysis

• Each subunit contains a carboxyl-terminal domain that is

homologous to the C1 and C2 domains of adenylyl cyclase;

alpha most closely resembles C1 while beta more closely resembles

C2.

Phosphodiesterases• PDEs are clinical targets for a range of biological disorders

such as retinal degeneration, congestive heart failure, depression, asthma, erectile dysfunction, and inflammation

• cAMP or cGMP + H2O 5’AMP or 5’GMP

• There are 11 families of enzymes and 21 genes– Families differ in substrate specificity

• cAMP: 4, 7, 8

• cGMP: 5, 6, 9

• Both: 1, 2, 3, 10, 11

– Families differ in regulation

– Families differ in tissue distribution

– PDE1, 3, 4, 6, 7, and 8 are multigene families

– PDE6 is expressed only in photoreceptors

Cyclic Nucleotide Phosphodiesterases• PDE3 is a target for CV drugs• PDE4 (cAMP) for depression and inflammation

– PDE4 is the chief PDE in inflammatory cells– Targeted diseases include atopic dermatitis, chronic obstructive

pulmonary disease (COPD, emphysema), and asthma– Side effects are common with PDE4 inhibitors such as rolipram

because of nausea and vomiting

• PDE5 for erectile dysfunction– Sildenafil (ViagraTM) was the first FDA approved PDE inhibitor

• A recent clinical trial indicates that Viagra is effective in the treatment of pulmonary hypertension– cGMP relaxes smooth muscle– Smooth muscle relaxation in arteries/arterioles decreased BP– Lungs have the highest activity of protein kinase G of any tissue

PDE5 inhibitors in the market• Forty million American men have some degree of erectile

dysfunction. • As many as 10 million have tried impotency drugs, but only about 3

million have stayed on the treatments.

• The drugs don’t save lives, but there’re big business. – Pfizer’s Viagra generated $894 million in sales, up 11% from the

prior year. – It is expected that the three approved PDE inhibitory drugs (next

slide) will generate $6 billion by 2009. – Cost $10-15 per pill

• There is some cross inhibition of PDE5 and PDE6 (rod cell) by Viagra which causes patients to see blue aberrations

• Patients taking nitroglycerin for ischemic coronary artery disease should not take PDE5 inhibitors because the combined effects of these agents might lower blood pressure to a lethal level– Nitroglycerin generates NO and activates soluble guanylyl cyclase

PDE Inhibitors in Clinical

Trials• Vigra

• Cialis Levitra

PDE5 Inhibitors for Erectile DysfunctionPfizer/Viagra

Blue pill

Bayer/Glaxo Levitra, Orange pill

Lilly/ICOS Cialis

Yellow pill

Generic name Sildenafil Vardenafil Tadalafil

Status Sold worldwide Sold worldwide Sold worldwide

Launch date 2000 2003 2003

Rate of successful intercourse (dose)

66% (20 mg) 75% (20 mg) 63% (20 mg)

Improved erections in nondiabetics

63% (25 mg) 85% (20 mg) 81% (20 mg)

Drug’s time to onset One hour before sex Within 15 min Within 30 min

Duration of effectiveness

Within four hours of taking the drug

Within 6 hours Up to 24 or more hours (TV ad says

36 h)

Food interaction Absorption reduced by a fatty meal

Absorption not affected by food

Absorption not affected by food

Nobel Prize in Medicine 1998for their discoveries concerning nitric oxide as a signaling

molecule in the cardiovascular system

http://www.nobel.se

Robert Louis FeridFurchgott Ignarro Murad

EDRF; endothelium derived relaxation factor

Second Messenger Summary

The End

• Otto Lowei: A drug is a substance when injected into an animal produces a paper

• Enzymology is fun

• Biochemistry is exhilarating