Molecular and Cellular Signaling - Martin Beckerman

328 13. Cell Fate and Polarity

Kalderon D [2002]. Similarities between the Hedgehog and Wnt signaling pathways.

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Ma D, et al. [2003]. Fidelity in planar cell polarity signaling. Nature, 421: 543–547. Mlodzik M [1999]. Planar polarity in the Drosophila eye: A multifaceted view of

signaling specificity and cross-talk. EMBO J., 18: 6873–6879.

Moon RT, Brown JD, and Torres M [1997]. Wnts modulate cell fate and behavior during vertebrate development. Trends Genet., 13: 157–162.

Wallingford JB, Fraser SE, and Harland RM [2002]. Convergent extension: The molecular control of polarized cell movement during embryonic development. Dev. Cell, 2: 695–706.

Hedgehog Signaling

Collins RT, and Cohen SM [2003].The secret life of Smoothened. Dev. Cell, 5: 823–824. Hammerschmidt M, Brook A, and McMahon AP [1997]. The world according to

Hedgehog. Trends Genet., 13: 14–21.

Ingham PW [1998]. Transducing Hedgehog: The story so far. EMBO J., 17: 3505–3511.

Ingham PW, and McMahon AP [2001]. Hedgehog signaling in animal development: Paradigms and principles. Genes Dev., 15: 3059–3087.

Taipale J, et al. [2002]. Patched acts catalytically to suppress the activity of Smoothened. Nature, 418: 892–897.

Morphogens

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Teleman AA, Strigini M, and Cohen SM [2001]. Shaping morphogen gradients. Cell, 105: 559–562.

Vertebrate Organizers and Body Plan

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Schier AF, and Shen MM [2000]. Nodal signaling in vertebrate development. Nature, 403: 385–389.

Sokol SY [1999]. Wnt signaling and dorsal-ventral axis specification in vertebrates.

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Feedback Loops in Development

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Freeman M [2000]. Feedback control of intercellular signalling in development. Nature, 408: 313–319.

Selector Genes, Gene Regulatory Networks, and the Segmentation Clock

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Davidson EH, et al. [2002]. A genomic regulatory network for development. Science, 295: 1669–1678.

Problems 329

Guss KA, et al. [2001]. Control of a genetic regulatory network by a selector gene. Science, 292: 1164–1167.

Hirata H, et al. [2002]. Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science, 298: 840–843.

Saga Y, and Takeda H [2001]. The making of the somite: Molecular events in vertebrate segmentation. Nature Rev. Genet., 2: 835–845.

Problems

13.1The core components and basic features of the four primary developmental pathways have now been introduced. The underlying picture is a fairly simple one in which ligand binding at the plasma membrane triggers a sequence of signaling events culminating in the activation of gene transcription in the nucleus. The signaling pathways possess a number of characteristics that enable them to guide the development of complex metazoans possessing a large variety of different cell types organized into tissues and organs. The first of these characteristics is that signaling is context dependent. The term “context” refers to the mix of proteins being expressed. How a cell responds to a signal depends on what other signaling events are taking place at the same time and in previous times that have altered the mix of proteins being expressed. How might context dependence come into play at the plasma membrane top influence signaling?

13.2Control points are situated at the plasma membrane, in the cytoplasm, and in the nucleus. Examples of cytoplasmic control points are the beta catenin complex formed in the Wnt signaling pathway and the Ci signaling complex formed in the Hedgehog signaling pathway. These along with MAP kinase and NF-kB modules function as major signaling nodes. Give some examples of how cellular context may influence what happens at these nodes in the signaling pathway.

13.3Two kinds of posttranslational modifications—phosphorylation and proteolysis are especially prominent in the four developmental pathways. These modifications operate individually and jointly in these pathways. List the various ways these modifications influence signaling.

13.4Despite the presence of different proteins, the developmental pathways have many features in common. In response to ligand binding, a series of signaling events takes place resulting in the translocation from the cytoplasm to the nucleus of a transcriptional factor. In these pathways downstream signaling elements can often function both as activators of transcription or as repressors, with the choice depending on cellular context. How does context come into play in the nucleus? How was this dual capability used in the pathways discussed in this chapter?

14

Cancer

Cancers arise from malfunctions in the cell control layer that lead to the unregulated proliferation of cells. The underlying causes of cancers are mutations and other alterations in DNA, and attendant inappropriate expression levels, of genes encoding proteins that either promote growth or restrain it, or direct the apoptosis machinery, or are responsible for DNA damage repair and signaling, and chromatin remodeling. The mutations may be heritable, that is, they may be present in the germ cells, or they may be produced in somatic cells.

DNA can be damaged in several ways. Hydrolytic processes and oxidative byproducts of normal cellular metabolism can damage DNA. Ionizing radiation from cosmic rays and from natural-occurring radioactive materials in the soil, water, and air such as uranium, thorium, and radon can damage DNA. In addition, ultraviolet (UV) radiation from the sun can damage DNA. The main step leading to DNA damage by environmental and endogenous stimuli is the generation of oxidative free radicals near the DNA. Free radicals are molecular species with unpaired electrons, making them highly reactive. The most damaging of these reactive oxidative species (ROS) is the hydroxyl radical. When produced in the vicinity of a DNA molecule the hydroxyl radicals and other ROS attack sugars and bases producing single strand breaks, base losses, and modified bases. In addition to this ROS mechanism, ionizing radiation such as X-rays and gamma irradiation can directly generate double strand breaks in DNA. When a cell that has been damaged divides without having the damage repaired the changes are carried on to its daughter cells and become a mutation.

In metastasis, malignant cancer cells break away from where they are immobilized, enter the circulatory system, and invade other organs. They form colonies, or secondary tumors, at the new locations and cause damage to their neighbors by appropriating their sources of nutrition. Cancers derived from epithelial cells are the most common type of cancer. In order for the cancerous epithelial cells to break away from their initial location they must detach from other epithelial cells and from the ECM. To do so they must disrupt the cell-to-cell adhesive contact established by E-

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FIGURE 14.1. Matrix metalloproteinase structure: Most MMPs contain an N- terminal signal peptide (not shown) that targets the enzyme for secretion, followed by a prodomain. The catalytic domain contains a zinc-binding domain and all MMPs except MMP7 and MMP26 contain a Hemopexin domain C-terminal to the catalytic domain and separated from it by a linker region. Dashed boxes represent domains present in some MMPs but not others. Included in this set are Furin-like domains, Type II Fibronectin repeats, and transmembrane + cytoplasmic segments.

cadherins, and cell-to-ECM contacts maintained by the integrins. Once they have detached from one another and from the ECM they have to pass through the basement membrane to reach and enter the circulatory system. This is accomplished using matrix metalloproteinases to degrade matrix proteins. The cancer cells make and secrete sufficient quantities of these proteolytic enzymes to weaken the membrane and allow passage of the cells through it into the blood vessels.

Matrix metalloproteinases (MMPs) are a family of over two dozen secreted proteolytic enzymes that (i) cleave components of the ECM, and (ii) cleave signaling proteins associated with the ECM and cell surfaces resulting in their activation and solubilization. These enzymes are normally synthesized in small quantities but production is increased in response to cytokines and stress, and accompanies the transformation of normal cells to cancerous ones.

As shown in Figure 14.1, MMPs contain a prodomain, which is cleaved to activate the enzyme, and fibronectin, hemopexin, and collagen V (not shown) domains that promote substrate and inhibitor binding. The furin domain provides an alternative cleavage site. Members of a family of four proteins called tissue inhibitors of metalloproteinases, or TIMPs, bind the hemopexin domain of MMPs, forming MMP-TIMP complexes that regulate the activities of the MMPs. A zinc-binding site is present in the catalytic domain. Zinc binding (mediated by a trio of histidine residues) is necessary, and this requirement gives rise to the name “metalloproteinase.”

14.1Several Critical Mutations Generate a Transformed Cell

The transformation of a normal cell into a cancerous one is a multistep process. Mutations accumulate over time and over cell generations, and the susceptibility to cancer increases rapidly with age. As the mutations accumulate a variety of genetic modifications are produced. These include alterations in chromosome number (aneuploidy) involving the loss or gain of

14.1 Several Critical Mutations Generate a Transformed Cell

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entire chromosomes, chromosome translocations that can generate a new gene by fusing two different genes, and gene duplications (gene amplification). One or more of these dramatic changes in chromosome organization are present in most human tumors.

The genetic targets of mutations that promote cancerous growth can be grouped into four functional classes. The first of these are genes that encode proteins that function in the growth signaling pathways. These gene products may convey progrowth signals or serve as brakes on growth. Mutated forms of the receptor tyrosine kinases, GTPases, and nonreceptor tyrosine kinases discussed in Chapters 10 and 11 are present in many different kinds of cancer. The developmental pathways discussed in the last chapter are not simply turned off at the end of embryogenesis, but rather remain active in one form or another during adult life. Mutated and/or overexpressed elements of these pathways are encountered in cancers as well.

The second set of mutations is to genes that encode proteins that regulate cell suicide. These target proteins either trigger or inhibit the cellular apoptosis program activated when aberrant conditions are detected. The third group of crucial mutations is to genes that encode cellular caretakers. These proteins carry out DNA repair and maintain chromosome integrity. The fourth and last set of crucial gene products consists of the central regulators of growth, repair, and death. These gene products are referred to as controller proteins. They are responsible for ensuring an orderly progression through the cell cycle, halting or advancing it when necessary and signaling to the apoptosis machinery when that outcome in required. Listed in Table 14.1 are a number of prominent members of each of these classes of proteins.

Oncoproteins are proteins that have been structurally altered by mutations in the genes that encode them. These proteins operate in the signal

TABLE 14.1. Protein with mutated and altered forms associated with cancer: Abbreviations—Oncoprotein (o); tumor suppressor (ts); DNA caretaker (c).

Protein	Function/pathway	Class	Cancer role
Ras	Growth	o	Many cancers
Src	Growth	o	Sarcomas
Abl	Growth	o	Leukemias
APC	Growth	ts	Colorectal cancers
b-catenin	Growth	o	Colorectal cancers
Myc	Growth	o	Many cancers
Bax	Apoptosis	ts	Many cancers
Bcl-2	Apoptosis	o	Many cancers
hMLH1	Repair	c	Colon cancers
hMSH2	Repair	c	Colon cancers
ATM	Repair	c	Ataxia telangiectasia
NBS	Repair	c	Nijmegen breakage syndrome
BRCA1,2	Repair	c	Breast/ovarian cancers
p53	Controller	ts	Most cancers
pRb	Controller	ts	Most cancers

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transduction, integration, and regulatory pathways involved in cellular growth, multiplication, differentiation, and death. As a result of the structural alterations these proteins do not function normally, but instead are changed in a manner that stimulates unregulated cell growth and proliferation thus promoting the development of cancer. Tumor suppressors are similar to oncoproteins except that they normally act as brakes on growth. When they suffer critical mutations these brakes on growth are removed.

14.2Ras Switch Sticks to “On” Under Certain Mutations

The first group of entries in Table 14.1 consists of proteins that relay growth signals from the plasma membrane to target sites in the cell interior. Ras and Src are prominent members of this group. Src is implicated in about 80% of human colon cancers. Ras oncoproteins are even more widespread in their cancer occurrences. They are present in 30 to 40% of human cancers. Not all mutations are equally important. Recall that a codon is a sequence of three nucleotide bases that encodes an amino acid. In Ras mutations, codons 12, 13, and 61 serve as hot spots for oncogene activity. The mutations occurring at these sites are point mutations that change one of the base pairs in the codons encoding glycine (12 and 13) and glutamine (61) into those encoding a different amino acid.

Ras is a crucial relay operating in the pathway that relays growth signals to downstream targets. It functions as a binary switch. Like all GTPases it is turned on by a GEF and turned off by a GAP. In the absence of growth signals, the switch is in its off position, but turns on in response to the appropriate signals. The mutations leading to cancer result in the switch being stuck in the on position, unable to turn off, and continually sending growth signals into the cell.

X-ray crystallography of Ras in complexes with its GEF (Figure 14.2) and GAP (Figure 14.3) provide insights into how the binary switch operates and how certain mutations leave Ras stuck in its on position. As can be seen in Figure 15.1a the Sos protein is organized into two domains, an N-terminal domain that is largely structural in nature, and a C-terminal that catalyzes the release of GDP. The catalytic domain forms a bowl about Ras. The Ras protein has two switch regions, called Sw 1 and Sw 2. These regions create a cavity within which GTP and GDP along with Mg2+ bind and release.

The Ras switch operates in the following manner. In the absence of growth signals, Ras is in its off position with GDP bound firmly in the pocket. When growth signals are present Sos is recruited to the plasma membrane and binds to the Ras-GDP complex. The aH helix of Sos engages the switches and flips Sw 1 to an open position in which it has rotated away from Sw 2. The GDP molecule dissociates from the complex, followed shortly thereafter by the dissociation of Sos. The GTP

14.2 Ras Switch Sticks to “On” Under Certain Mutations

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FIGURE 14.2. Structure of Ras in a complex with its Sos GEF as determined using X-ray crystallography: Ras is shown in light gray while Sos is depicted in black. Small filled circles and squares superimposed on the figure serve to outline Sw 1 and Sw 2, respectively. The figure was generated using Protein Explorer with atomic coordinates deposited in the Brookhaven Protein Data Bank (PDB) under accession code 1BKD.

FIGURE 14.3. Structure of Ras in a complex with its GAP as determined using X- ray crystallography: Ras is shown in light gray while the RasGAP is depicted in black. Small filled circles and squares superimposed on the figure serve to outline Sw 1 and Sw 2, respectively. The Gly12 residue is located at the tip of the Ras P- loop while a second crucial residue, Glu61, located in the Ras Sw 2 region in close opposition to the arginine finger loop of the RasGAP. The figure was generated using Protein Explorer with atomic coordinates deposited in the Brookhaven PDB under accession code 1WQ1.

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molecule is plentiful and binds in the pocket that was vacated by the GDP molecule.

Mutations of the glycine residue at position 12 in the Ras chain convert Ras into a form that is active all the time.That is, the RasGAP is unable to turn off Ras.The reason for this can be discerned from the crystal structure exhibited in Figure 14.3. The Gly12 residue is positioned at a crucial place at the very end of the P loop.Because of its small size,replacement of glycine by any other residue blocks the arginine in the finger from interacting with the ATP molecule bound in the cleft, and hydrolysis is consequently impeded.

14.3Crucial Regulatory Sequence Missing in Oncogenic Forms of Src

Both Src and Abl are nonreceptor tyrosine kinases and were discussed in Chapter 11. The Src gene was first identified in retroviruses. These are viruses that use RNA as their genetic coding medium rather than DNA. When a retrovirus invades a cell the viral RNA is transformed into DNA and integrated into the host genome. Retroviruses sometimes acquire genes with oncogenic capabilities from an early host and deliver these into later hosts. These genes are referred to viral oncogenes. The Src oncogene was first identified in the chicken Rous sarcoma virus, and then a cellular counterpart to the viral oncogene was found in normal cells in the chicken and then in humans. The viral forms of Src and other viral oncogenes usually differ in some way from their cellular counterparts. For that reason the viral form of Src is denoted as v-Src while its cellular form is designated as c-Src, and a similar situation obtains for other oncogenes.

Recall from Chapter 11 that a critical residue Tyr527 located in its COOH tail controls the catalytic activity of c-Src. Phosphorylation of this residue by Csk deactivates c-Src, and the Tyr527 phosphorylation site is required for proper function of the kinase. In v-Src, the tail region containing Tyr527 is missing, and the truncated protein cannot be turned off. The result is that the v-Src protein is constitutively active sending uncontrolled cell growth and proliferation signals to the nucleus. The cellular form of Src can be mutated in several ways to generate oncogenic forms. Point mutations in the codon for Tyr527 converting this amino acid to phenylalanine can transform Src as can specific mutations that disrupt the ability of the SH2 and SH3 domains to cooperate with the COOH tail in inhibiting Src activation.

14.4Overexpressed GFRs Spontaneously Dimerize in Many Cancers

Growth factors and growth factor receptors (GFRs) support the development of malignant tumors in several ways. One prominent contributor to the onset of malignancy is the vascular endothelial growth factor (VEGF).

14.5 GFRs & Adhesion Molecules Cooperate to Promote Tumor Growth

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In order for a solid tumor to grow and thrive it must have an adequate blood supply. In response to this need for vascular expansion, tumor angiogenesis takes place. The expression of messenger RNAs for VEGF ligands is enhanced in most human tumor cells. Increased VEGF mRNAs are present in rapidly growing glioblastoma multiform brain tumors, and in cancers of the lung, breast, gastrointestinal tract, female reproductive organs, thyroid gland, and urinary tract.

Growth factor receptor dimerization is a critical step in relaying signals conveyed by polypeptide growth factors into the cell interior.As discussed in Chapter 11, receptor tyrosine kinases are brought into close physical proximity through ligand binding, resulting in the formation of receptor dimers or oligomers. Autophosphorylation in the activation loop occurs next followed by recruitment of cytoplasmic signaling molecules. In the absence of a ligand the receptors do not dimerize and there is no signaling across the plasma membrane. In contrast to this normal situation, spontaneous dimerization occurs in many cancers. In these abnormal situations the receptors dimerize in the absence of ligand binding. Ligand-free dimerization can be produced in several different ways. Most often it is generated through overexpression of receptors arising as a consequence of gene amplification. It can also be generated through point mutations and exon deletions.

Epidermal growth factor receptors (EGFRs) undergo spontaneous dimerization in many cancers including those of the breast, lung and ovarian cancers and gliomas, and brain tumors of glial origin. Amplification of the EGFR (ErbB1) gene occurs in about 40% of gliomas, and the amplification of the ErbB2 gene takes place in about 30% of breast cancers. In many of the brain tumors, gene rearrangements accompany gene amplification and these alterations often involving truncations of portions of the molecule. The main effect of spontaneous dimerization is to activate a pathway that sends inappropriate growth/proliferation signals to the nucleus.

14.5GFRs and Adhesion Molecules Cooperate to Promote Tumor Growth

Alterations in the mix of cell adhesion molecules being expressed accompany tumor progression. The altered expression patterns occur not only during metastasis but also during solid tumor growth. Most cancers develop from epithelial cells, and loss of E-cadherins is a common occurrence. Recall that E-cadherins help maintain tight adhesive contacts in populations of these cells. Loss of adhesive junctions and changes in cytoskeleton organization accompanies the transformation to malignancy. Among the changes in expression patterns are the upregulation of aVb3 and a6b4 integrins, and the switching from N-cadherins to E-cadherins and back when adhesive contacts are again needed.

Integrins along with cadherins and Ig superfamily cell adhesion molecules form complexes with growth factor receptors. Examples of coopera-

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tivity between adhesion and growth factor receptors are a6b1 and a6b4 and EGFRs, NCAM, and N-cadherins with FGFRs, and VE-cadherins with VEGF receptors. One result of this form of association is the ability of integrins and/or growth factor receptors to convey signals into the cell without having to engage their natural ligands. Clustering brings the receptors into close proximity with one another, and promotes phosphorylation and the recruitment of cytoplasmic signaling transducers, thereby alleviating the need for ligand engagement.

A second consequence of the growth factor receptor–adhesion molecule clustering is the strengthening of signals that would otherwise be too weak to elicit a cellular response if conveyed by one or the other alone. An example of this form of cooperativity is that which occurs between Met and a6b4 integrins. Recall from Chapter 10 that Met is the receptor for HGF/SF, a set of diffusible ligands that are central participants in invasive growth and branching morphogenesis. SF does not stimulate growth, but rather triggers the dissociation, or scattering, of cells. By forming clusters the cytoplasmic segments of the Met receptors and integrins come into close contact; they are able to promote phosphorylation and provide multiple docking sites for adapters and other cytoplasmic signaling elements. In this second signaling role, the a6b4 acts as an amplifier to increase the magnitude of the cellular response to a growth signal and promote invasive growth independent of binding to the ECM.

14.6Role of Mutated Forms of Proteins in Cancer Development

The likelihood of getting a colorectal tumor exceeds 50% by age 70. Most of these tumors do not progress to a lethal stage, but nevertheless colorectal tumors are the second leading cause of cancer death in the United States.

Mutated forms of several gene products contribute to the onset of colorectal tumors. Two of these, the APC protein and b-catenin will be discussed in this section,while mutations in another pair of gene products,the mismatch repair proteins hMLH1 and hMSH2, will be examined in a later section.

The adenomatous polyposis coli (APC) protein and b-catenin participate in the Wnt signaling pathway. The Wnt signaling system is thought of as a developmental pathway, and was discussed in the last chapter. APC is localized in the basolateral compartment of epithelial cells along with glycogen synthase kinase (GSK) that regulates its signaling activity. Recall that in the absence of a Wnt signal, GSK phosphorylates b-catenin thereby tagging that molecule for destruction. In the presence of Wnt signaling GSK is antagonized and does not tag b-catenin. The latter is stabilized as a cytoplasmic monomer and can then translocate to the nucleus. APC forms a complex with GSK and b-catenin. If APC is absent or is mutated, b-catenin is not properly regulated by GSK. Instead, the b-catenin levels are raised