Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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Bcr-Abl that consists primarily of sequences for the c-Abl kinase. However, the bcr-abl gene is not regulated

appropriately; it is expressed at higher levels than the gene encoding the normal c-Abl kinase. In addition, the Bcr-Abl

protein may have regulatory properties that are subtly different from those of the c-Abl kinase itself. Thus, leukemia

cells express a unique target for chemotherapy. Recent clinical trials of a specific inhibitor of the Bcr-Abl kinase have

shown dramatic results; more than 90% of patients responded well to the treatment. This approach to cancer

chemotherapy is fundamentally distinct from most approaches, which target cancer cells solely on the basis of their rapid

growth, leading to side effects because normal rapidly growing cells also are affected. Thus, our understanding of signal-

transduction pathways is leading to conceptually new disease treatments.

15.5.2. Cholera and Whooping Cough Are Due to Altered G-Protein Activity

We consider here some pathologies of the G-protein-dependent signal pathways. Let us first consider the

mechanism of action of the cholera toxin, secreted by the intestinal bacterium Vibrio cholera. Cholera is an acute

diarrheal disease that can be life threatening. It causes voluminous secretion of electrolytes and fluids from the intestines

of infected persons. The cholera toxin, choleragen, is a protein composed of two functional units a B subunit that

binds to G

gangliosides of the intestinal epithelium and a catalytic A subunit that enters the cell. The A subunit

catalyzes the covalent modification of a G

protein: the α subunit is modified by the attachment of an ADP-ribose to an

arginine residue. This modification stabilizes the GTP-bound form of G

, trapping the molecule in the active

conformation. The active G protein, in turn, continuously activates protein kinase A. PKA opens a chloride channel (a

CFTR channel; Section 13.3) and inhibits the Na

-H

exchanger by phosphorylation. The net result of the

phosphorylation of these channels is an excessive loss of NaCl and the loss of large amounts of water into the intestine.

Patients suffering from cholera for 4 to 6 days may pass as much as twice their body weight in fluid. Treatment consists

of rehydration with a glucose-electrolyte solution.

Whereas cholera is a result of a G protein trapped in the active conformation, causing the signal-transduction pathway to

be perpetually stimulated, pertussis, or whooping cough, is a result of the opposite situation. Pertussis toxin also adds an

ADP-ribose moiety,

in this case, to a G

protein, a G

protein that inhibits adenyl cyclase, closes Ca

channels, and

opens K

channels. The effect of this modification, however, is to lower the G protein's affinity for GTP, effectively

trapping it in the "off" conformation. The pulmonary symptoms have not yet been traced to a particular target of the G

protein. Pertussis toxin is secreted by Bordetella pertussis, the bacterium responsible for whooping cough.

Cholera and pertussis are but two examples of diseases caused by defects in G proteins. Table 15.4 lists others. In light

of the fact that G proteins relay signals for more than 500 receptors, it is likely that this list will continue to grow.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism 15.5. Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

Figure 15.35. Src Structure.

(A) Cellular Src includes an SH3 domain, an SH2 domain, a protein kinase domain, and a

carboxyl-terminal tail that includes a key tyrosine residue. (B) Structure of c-Src in an inactivated form with the

key tyrosine residue phosphorylated. The phosphotyrosine residue is bound in the SH2 domain; the linker between

the SH2 domain and the protein kinase domain is bound by the SH3 domain. These interactions hold the kinase domain

in an inactive conformation.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism 15.5. Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

Figure 15.36. Activation Pathways for c-Src. Inactive c-Src can be activated by one of at least three distinct pathways:

(1) displacement of the SH2 domain, (2) dephosphorylation, or (3) displacement of the SH3 domain.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism 15.5. Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

Figure 15.37. Formation of the Bcr-Abl Gene by Translocation. In chronic myologenous leukemia, parts of

chromosomes 9 and 22 are reciprocally exchanged, causing the bcr and abl genes to fuse. The protein kinase encoded by

the bcr-abl gene is expressed at higher levels in cells having this translocation than is the c-abl gene in normal cells.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism 15.5. Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

Table 15.4. Diseases of heterotrimeric G proteins

Disease

Excessive signaling

Cholera

Cancer (adenoma) of pituitary and thyroid

Cancer (adenoma) of adrenal and ovary

Essential hypertension

Deficient signaling

Night blindness

Pseudohypoparathyroidism type Ib

Pertussis

Source: After Z. Farfel, H. R. Bourne, and T. Iiri. N. Engl. J. Med. 340(1999):1012.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism

15.6. Recurring Features of Signal-Transduction Pathways Reveal Evolutionary

Relationships

Many features of signal-transduction pathways are ancient. For example, cAMP signals the need for energy in

prokaryotes as well as eukaryotes, although the mechanisms for detecting cAMP are different. Similarly, the GTP-

binding proteins the G

subunits of the hetero-trimeric G proteins and the members of the Ras family are part of an

ancient superfamily of evolutionarily related proteins. Other members of this superfamily are proteins that cycle between

ATP- and ADP-bound forms; these proteins function in ATP synthesis (Section 18.4.5) and in generating molecular

motion (Chapter 34). The superfamily also includes proteins taking part in protein synthesis (Section 29.4.2). The key

feature of these proteins is that they undergo significant conformational changes on binding nucleoside triphosphates and

hydrolyzing them to nucleoside diphosphates. These proteins can thus function as molecular "on-off" switches. A

domain with this ability must have arisen early in evolution and been adapted to meet a range of biochemical needs since.

Other proteins crucial to signal-transduction pathways arose much later. For example, the eukaryotic protein kinases are

one of the largest protein families in all eukaryotes and yet appear to be absent in prokaryotes. The evolution of the

eukaryotic protein kinase domain appears to have been an important biochemical step in the appearance of eukaryotes

and the subsequent development of multicellular organisms.

Entire signaling pathways have been conserved between organisms. For example, a key pathway in eye development in

Drosophila is completely analogous to the EGF pathways in human beings (Figure 15.38). Thus, the wiring of this

growth-control pathway is at least 800 million years old.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism 15.6. Recurring Features of Signal-Transduction Pathways Reveal Evolutionary Relationships

Figure 15.38. Pathway Conservation. A pathway homologous to the mammalian EGF signal-transduction pathway

functions in Drosophila to control the development of a specific photoreceptor cell in the eye.

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism

Summary

The highly specific binding of signal molecules, many of which are hormones and growth factors, to receptor molecules

initiates the signal-transduction cascade. Secondary messengers carry the signal inside the cell and often use protein

phosphorylation as a signaling device.

Seven-Transmembrane-Helix Receptors Change Conformation in Response to Ligand

Binding and Activate G Proteins

Seven-transmembrane-helix receptors operate in conjunction with heterotrimeric G proteins. The binding of hormone to

a 7TM receptor triggers the exchange of GTP for GDP bound to the α subunit of the G protein. G

proteins can transmit

information in a number of ways. G

-GTP activates adenylate cyclase, an integral membrane protein that catalyzes the

synthesis of cAMP. Cyclic AMP then activates protein kinase A by binding to its regulatory subunit, thus unleashing its

catalytic chains. PKA, a multifunctional kinase, alters the activity of many target proteins by phosphorylating serine and

threonine residues.

The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates

Two Messengers

The phosphoinositide cascade is mediated by 7TM receptors and G

proteins. The receptor-triggered activation of

phospholipase C generates two intracellular messengers by hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Inositol

trisphosphate opens calcium channels in the endoplasmic and sarcoplasmic reticulum membranes, leading to an elevated

level of Ca

in the cytosol. Diacylglycerol activates protein kinase C, which phosphorylates serine and threonine

residues in target proteins.

Calcium Ion Is a Ubiquitous Cytosolic Messenger

Calcium ion acts by binding to calmodulin and other calcium sensors. Calmodulin contains four calcium-binding

modules called EF hands that recur in other proteins. Ca

-calmodulin activates target proteins by binding to positively

charged amphipathic helices.

Some Receptors Dimerize in Response to Ligand Binding and Signal by Cross-

Phosphorylation

Some ligands induce dimerization of the receptors to which they bind. Such a receptor contains an extracellular domain

that binds the ligand, a transmembrane region, and a cytosolic domain that either binds or contains a protein kinase. The

growth-hormone receptor participates in an example of this type of signal-transduction pathway. Dimerization of the

receptor activates Janus kinase 2, a protein kinase associated with the intracellular part of the receptor. The kinase, in

turn, phosphorylates and activates a transcription factor called STAT5.

Intrinsic tyrosine kinases are covalently incorporated in the intracellular domains of some receptors, such as epidermal

growth factor receptor and the insulin receptor. When such receptor tyrosine kinases dimerize, cross-phosphorylation

occurs. The phosphorylated tyrosines in activated receptor tyrosine kinases serve as docking sites for SH2 domains

present in numerous signaling proteins and permit further propagation of the signal. A prominent component of such

pathways is the small GTPase Ras. The Ras protein, like the G

subunit, cycles between an inactive form bound to GDP

and an active form bound to GTP.

Defects in Signaling Pathways Can Lead to Cancer and Other Diseases

If the genes encoding components of the signal-transduction pathways are altered by mutation, pathological conditions,

most notably cancer, may result. In their mutated form, these genes are called oncogenes. The normal counterparts are

called proto-oncogenes and function in pathways that control cell growth and replication. Mutated versions of ras are

frequently found in human cancers.

Recurring Features of Signal-Transduction Pathways Reveal Evolutionary

Relationships

Many aspects of signal transduction are ancient and appear throughout the kingdoms of life. G proteins of various classes

are employed as molecular switches in a host of biochemical processes.

Key Terms

ligand

primary messenger

second messenger

protein kinase

protein phosphatase

seven-transmembrane-helix (7TM) receptor

G protein

adenylate cyclase

G-protein-coupled receptor (GPCR)

desensitization (adaptation)

protein kinase A (PKA)

phosphoinositide cascade

phospholipase C

protein kinase C (PKC)

pseudosubstrate sequence

calmodulin (CaM)

EF hand

calmodulin-dependent protein (CaM) kinase

SH2 domain

receptor tyrosine kinase (RTK)

SH3 domain

small G protein

oncogene

proto-oncogene

II. Transducing and Storing Energy 15. Signal-Transduction Pathways: An Introduction to Information Metabolism

Problems

Levels of amplification. At which stages in the signaling pathway from epinephrine to cAMP does a significant

amount of amplification occur? Answer the same question for the signaling pathways from human growth hormone

to STAT5 and from EGF to Ras.

See answer

Active mutants. Some protein kinases are inactive unless they are phosphorylated on key serine or threonine

residues. In some cases, active enzymes can be generated by mutating these serine or threonine residues to

glutamate. Propose an explanation.

See answer

In the pocket. SH2 domains bind phosphotyrosine residues in deep pockets on their surfaces. Would you expect SH2

domains to bind phosphoserine or phosphothreonine with high affinity? Why or why not?

See answer

Inhibitory constraints. Many proteins in signal-transduction pathways are activated by the removal of an inhibitory

constraint. Give two examples of this recurring mechanism.

See answer

Pseudosubstrate mutant. A mutated form of a protein kinase C isozyme that has three changes in amino acids in the

pseudosubstrate sequence is isolated. What properties would you expect this mutated form to have?

See answer

Antibodies mimicking hormones. Antibodies have two identical antigen-binding sites. Remarkably, antibodies to the

extracellular parts of growth-factor receptors often lead to the same cellular effects as does exposure to growth

factors. Explain this observation.

See answer

Facile exchange. A mutated form of the α subunit of the heterotrimeric G protein has been identified; this form

readily exchanges nucleotides even in the absence of an activated receptor. What effect would you expect this

mutated α subunit to have on its signaling pathway?

See answer

Blocking one side. Human growth hormone binds simultaneously to two growth-hormone receptors through two

different surfaces. Suppose sequence changes in a mutated form of growth hormone disrupt one interaction surface

but do not affect the other. What properties would such a mutated hormone have? Why might it be useful?

See answer

Diffusion rates. Normally, rates of diffusion vary inversely with molecular weights; so smaller molecules diffuse

faster than do larger ones. In cells, however, calcium ion diffuses more slowly than does cAMP. Propose a possible

explanation.

See answer

10.

Awash with glucose. Glucose is mobilized for ATP generation in muscle in response to epinephrine, which

activates G

. Cyclic AMP phosphodiesterase is an enzyme that converts cAMP into AMP. How would inhibitors

of cAMP phosphodiesterase affect glucose mobilization in muscle?

See answer

Chapter Integration Problem

11.

Nerve growth factor pathway. Nerve growth factor (NGF) binds to a protein tyrosine kinase receptor. The amount

of diacylglycerol in the plasma membrane increases in cells expressing this receptor when treated with NGF.

Propose a simple signaling pathway and identify the isoform of any participating enzymes. Would you expect the

concentrations of any other common second messengers to increase on NGF treatment?

See answer

Mechanism Problem

12.

Distant relatives. The structure of adenylate cyclase is similar to the structures of some types of DNA polymerases,

suggesting that these enzymes derived from a common ancestor. Compare the reactions catalyzed by these two

enzymes. In what ways are they similar?

See answer

Data Interpretation Problems

13.

Establishing specificity. You wish to determine the hormone-binding specificity of a newly identified membrane

receptor. Three different hormones, X, Y, and Z, were mixed with the receptor in separate experiments, and the

percentage of binding capacity of the receptor was determined as a function of hormone concentration, as shown in

graph A.

(a) What concentrations of each hormone yield 50% maximal binding?

(b) Which hormone shows the highest binding affinity for the receptor?

You next wish to determine whether the hormone-receptor complex stimulates the adenylate cyclase cascade. To do

so, you measure adenylate cyclase activity as a function of hormone concentration, as shown in graph B.

hormone to enhance adenylate cyclase activity? What can you conclude about the mechanism of action of the

hormone-receptor complex?

(d) Suggest experiments that would determine whether a G

protein is a component of the signal-transduction

pathway.

See answer

14.

Binding issues. A scientist wishes to determine the number of receptors specific for a ligand X, which he has in

both radioactive and nonradioactive form. In one experiment, he adds increasing amounts of the radioactive X and

measures how much of it is bound to the cells. The result is shown as total activity in the adjoining graph. Next, he

performs the same experiment, except that he includes a several hundredfold excess of nonradioactive X. This

result is shown as nonspecific binding. The difference between the two curves is the specific binding.

(a) Why is the total binding not an accurate representation of the number of receptors on the cell surface?

(b) What is the purpose of performing the experiment in the presence of excess nonradioactive ligand?

See answer

15.

Counting receptors. With the use of experiments such as those described in problems 13 and 14, it is possible to

calculate the number of receptors in the cell membrane. Suppose that the specific activity of the ligand is 10

cpm

per millimole and that the maximal specific binding is 10

cpm per milligram of membrane protein. There are 10

cells per milligram of membrane protein. Assume that one ligand binds per receptor. Calculate the number of

receptor molecules present per cell.

See answer

Media Problem

16.

Grubby binding. The Structural Insights module on SH2 domains describes some of the determinants of

SH2 specificity and the ways in which SH2-phosphotyrosine binding can affect protein function. Given that

the Src kinase SH2 domain binds Src phosphotyrosine 527, what effect do you think mutation of Glu 529 to Asn

would have on the protein kinase activity of Src? Suppose you now obtained a second mutation within Src that

reversed the effect of the first. Can you predict what that second mutation might be?