Protein Kinases, the Most Important Biochemical Regulatory System in Animal Cells Big Picture

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

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

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

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

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

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

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 Ligands Transforming 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

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 Nomenclature Mitogen 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

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 Phosphatases

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 Dysfunction

Nobel Prize in Medicine 1998

• for their discoveries concerning nitric oxide as a signaling molecule in the cardiovascular system

• http://www.nobel.se

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