The need for intercellular signaling is never greater than during animal development. Each cell in the embryo has to be guided along one developmental pathway or another according to its history, its position, and the character of its neighbors. At each step in the pathway, it must exchange signals with its neighbors to coordinate its behavior with theirs. Most of the signaling pathways already discussed are widely used for these purposes. But there are also others that relay signals in other ways from cell-surface receptors to the interior of the cell. These additional signaling pathways all depend, in part at least, on regulated proteolysis. Although most of them first came to light through genetic studies in Drosophila, they have been highly conserved in evolution and are used over and over again during animal development. As we discuss in Chapter 22, they also have a crucial role in the many developmental processes that continue in adult tissues.
We discuss four of these signaling pathways in this section: the pathway mediated by the receptor protein Notch, the pathway activated by secreted Wnt proteins, the pathway activated by secreted Hedgehog proteins, and the pathway that depends on activation of the latent gene regulatory protein NF-κB. All of these pathways have crucial roles in animal development. If any one of them is inactivated in a mouse, for example, development is seriously disturbed, and the mouse dies as an embryo or at birth. (We discuss the roles of Notch, Wnt, and Hedgehog signaling in embryonic development in Chapter 21.)
Signaling through the Notch receptor protein may be the most widely used signaling pathway in animal development. As discussed in Chapter 21, it has a general role in controlling cell fate choices during development, mainly by amplifying and consolidating molecular differences between adjacent cells. Although Notch signaling is involved in the development of most tissues, it is best known for its role in nerve cell production in Drosophila. The nerve cells usually arise as isolated single cells within an epithelial sheet of precursor cells. During the process, each future nerve cell or committed nerve-cell precursor signals to its immediate neighbors not to develop in the same way at the same time, a process known as lateral inhibition. In a fly embryo, for example, the inhibited cells around the future nerve-cell precursors develop into epidermal cells. Lateral inhibition depends on a contact-dependent signaling mechanism that is mediated by a signal protein called Delta, displayed on the surface of the future neural cell. By binding to Notch on a neighboring cell, Delta signals to the neighbor not to become neural (Figure 15-70). When this signaling process is defective in flies, the neighbors of neural cells also develop as neural cells, producing a huge excess of neurons at the expense of epidermal cells, which is lethal. Signaling between adjacent cells via Notch and Delta (or the Deltalike ligand Serrate) regulates cell fate choices in a wide variety of tissues and animals, helping to create fine-grained patterns of distinct cell types. The Notch-mediated signal can have other effects beside lateral inhibition; in some tissues, for example, it works in the opposite way, causing neighboring cells to behave similarly.
Lateral inhibition mediated by Notch and Delta during nerve cell development in Drosophila. When individual cells in the epithelium begin to develop as neural cells, they signal to their neighbors not to do the same. This inhibitory, contact-dependent (more...)
Both Notch and Delta are single-pass transmembrane proteins, and both require proteolytic processing to function. Although it is still unclear why Delta has to be cleaved, the cleavage of Notch is central to how Notch activation altersgene expression in the nucleus. When activated by the binding of Delta on another cell, an intracellular protease cleaves off the cytoplasmic tail of Notch, and the released tail moves into the nucleus to activate the transcription of a set of Notch-response genes. The Notch tail acts by binding to a gene regulatory protein called CSL (so named because it is called CBF1 in mammals, Suppressor of Hairless in flies, and Lag-1 in worms); this converts CSL from a transcriptional repressor into a transcriptional activator. The products of the main genes directly activated by Notch signaling are themselves gene regulatory proteins, but with an inhibitory action: they block the expression of genes required for neural differentiation (in the nervous system), and of various other genes in other tissues.
The Notch receptor undergoes three proteolytic cleavages, but only the last two depend on Delta. As part of its normal biosynthesis, a protease called furin acts in the Golgi apparatus to cleave the newly synthesized Notch protein in its future extracellular domain. This cleavage converts Notch into a heterodimer, which is then transported to the cell surface as the mature receptor. The binding of Delta to Notch induces a second cleavage in the extracellular domain, mediated by a different protease. A final cleavage quickly follows, cutting free the cytoplasmic tail of the activated receptor (Figure 15-71).
The processing and activation of Notch by proteolytic cleavage. The numbered red arrowheads indicate the sites of proteolytic cleavage. The first proteolytic processing step occurs within the trans Golgi network to generate the mature heterodimeric Notch (more...)
The cleavage of the Notch tail occurs very close to the plasma membrane, just within the transmembrane segment. In this respect it resembles the cleavage of another, more sinister transmembrane protein--the β-amyloid precursor protein (APP), which is expressed in neurons and is implicated in Alzheimer's disease. APP is cleaved within its transmembrane segment, releasing one peptide fragment into the extracellular space of the brain and another into thecytosol of the neuron. In Alzheimer's disease, the extracellular fragments accumulate in excessive amounts and aggregate into filaments that form amyloid plaques, which are believed to injure nerve cells and contribute to their loss. The most frequent genetic cause of early-onset Alzheimer's disease is a mutation in the presenilin-1 (PS-1) gene, which encodes an 8-pass transmembrane protein that participates in the cleavage of APP. The mutations in PS-1 cause cleavage of APP into amyloid-plaque-forming fragments at an increased rate. Genetic evidence in C. elegans, Drosophila, and mice indicates that the PS-1 protein is a required component of the Notch signaling pathway, helping to perform the final cleavage that activates Notch. Indeed, Notch signaling and cleavage are greatly impaired in PS-1-deficient cells.
Remarkably, Notch signaling is regulated by glycosylation. The Fringe family of glycosyltransferases adds extra sugars to the O-linked oligosaccharide (discussed in Chapter 13) on Notch, which alters the specificity of Notch for its ligands. This has provided the first example of the modulation of ligand-receptor signaling by differential receptor glycosylation.
Wnt proteins are secreted signal molecules that act as local mediators to control many aspects of development in all animals that have been studied. They were discovered independently in flies and in mice: in Drosophila, the wingless (wg) gene originally came to light because of its role in wing development, while in mice, the Int-1 gene was found because it promoted the development of breast tumors when activated by the integration of a virus next to it. The cell-surface receptors for the Wnts belong to the Frizzled family of seven-pass transmembrane proteins. They resemble G-protein-linked receptors in structure, and some of them can signal through G proteins and the inositol phospholipidpathway discussed earlier. They mainly signal, however, through G-protein-independent pathways, which require a cytoplasmic signaling protein called Dishevelled.
The best characterized of the Dishevelled-dependent pathways acts by regulating the proteolysis of a multifunctionalprotein called β-catenin (or Armadillo in flies), which functions both in cell-cell adhesion and as a latent gene regulatory protein. Wnts activate this pathway by binding to both a Frizzled protein and a co-receptor protein. The co-receptor protein is related to the low density lipoprotein (LDL) receptor protein (discussed in Chapter 13) and is therefore called LDL-receptor-related protein (LRP). It is uncertain how Frizzled and LRP activate Dishevelled, which relays the signal onward.
In the absence of Wnt signaling, most of a cell's β-catenin is located at cell-cell adherens junctions, where it is associated with cadherins, which are transmembrane adhesion proteins. As discussed in Chapter 19, the β-catenin in these junctions helps link the cadherins to the actin cytoskeleton. Any β-catenin not associated with cadherins is rapidly degraded in the cytoplasm. This degradation depends on a large degradation complex, which recruits β-catenin and contains at least three other proteins (Figure 15-72A):
A model for the Wnt activation of the β-catenin signaling pathway. (A) In the absence of a Wnt signal, some β-catenin is bound to the cytosolic tail of cadherin proteins (not shown) and any cytosolic β-catenin becomes bound by(more...)
A serine/threonine kinase called glycogen synthase kinase-3β (GSK-3β) phosphorylates β-catenin, thereby marking the protein for ubiquitylation and rapid degradation in proteasomes.
The tumor-suppressor protein adenomatous polyposis coli (APC) is so named because the gene encoding it is often mutated in a type of benign tumor (adenoma) of the colon. The tumor projects into the lumen as a polyp, which can eventually become malignant. APC helps promote the degradation of β-catenin by increasing the affinity of the degradation complex for β-catenin, as required for effective phosphorylation of β-catenin by GSK-3β.
A scaffold protein called axin holds the protein complex together.
The binding of a Wnt protein to Frizzled and LRP leads to the inhibition of β-catenin phosphorylation and degradation. The mechanism is not understood in detail, but it requires Dishevelled and several other signaling proteins that bind to Dishevelled, including the serine/threonine kinase called casein kinase 1. As a result, unphosphorylated β-catenin accumulates in the cytoplasm and nucleus (Figure 15-72B).
In the nucleus, the target genes for Wnt signaling are normally kept silent by an inhibitory complex of generegulatory proteins, which includes proteins of the LEF-1/TCF family bound to the corepressor protein Groucho (seeFigure 15-72A). The increase in undegraded β-catenin caused by Wnt signaling allows β-catenin to enter the nucleus and bind to LEF-1/TCF, displacing Groucho. The β-catenin now functions as a coactivator, inducing the transcription of the Wnt target genes (see Figure 15-72B).
Among the genes activated by β-catenin is c-myc, which encodes a protein (c-Myc) that is a powerful stimulator of cell growth and proliferation (discussed in Chapter 17). Mutations of the APC gene occur in 80% of human colon cancers. These mutations inhibit the protein's ability to bind β-catenin, so that β-catenin accumulates in the nucleusand stimulates the transcription of c-myc and other Wnt target genes, even in the absence of Wnt signaling. The resulting uncontrolled cell proliferation promotes the development of cancer.
Like Wnt proteins, the Hedgehog proteins are a family of secreted signal molecules that act as local mediators in many developmental processes in both invertebrates and vertebrates. Abnormalities in the Hedgehog pathway duringdevelopment can be lethal and in adult cells can also lead to cancer. The Hedgehog proteins were discovered inDrosophila, where a mutation in the only gene encoding such a protein produces a larva with spiky processes (denticles) resembling a hedgehog. At least three genes encode Hedgehog proteins in vertebrates--sonic, desert, andindian hedgehog. The active form of all Hedgehog proteins is unusual in that it is covalently coupled to cholesterol, which helps to restrict its diffusion following secretion. The cholesterol is added during a remarkable processing step, in which the protein cleaves itself. The proteins are also modified by the addition of a fatty acid chain, which, for unknown reasons, can be required for their signaling activity.
Two transmembrane proteins, Patched and Smoothened, mediate the responses to all Hedgehog proteins. Patched is predicted to cross the plasma membrane 12 times, and it is the receptor that binds the Hedgehog protein. In the absence of a Hedgehog signal, Patched inhibits the activity of Smoothened, which is a 7-pass transmembrane proteinwith a structure similar to a Frizzled protein. This inhibition is relieved when a Hedgehog protein binds to Patched, allowing Smoothened to relay the signal into the cell. Most of what we know about the downstream signaling pathway activated by Smoothened comes from genetic studies in flies, and it is the fly pathway that we summarize here.
In some respects the Hedgehog signaling pathway in Drosophila operates similarly to the Wnt pathway. In the absence of a Hedgehog signal, a gene regulatory protein called Cubitus interruptus (Ci) is proteolytically cleaved in proteasomes. Instead of being completely degraded, however, it is processed to form a smaller protein that accumulates in the nucleus, where it acts as a transcriptional repressor, helping to keep some Hedgehog-responsive genes silent. The proteolytic processing of the Ci protein depends on a large multiprotein complex. The complex contains a serine/threonine kinase (called Fused) of unknown function, an anchoring protein (called Costal) that binds the complex to microtubules (keeping Ci out of the nucleus), and an adaptor protein (called Suppressor of Fused) (Figure 15-73A). When Hedgehog binds to Patched to activate the signaling pathway, Ci processing is suppressed, and the unprocessed Ci protein is released from its complex and enters the nucleus, where it activates the transcription of Hedgehog target genes (Figure 15-73B).
A model for Hedgehog signaling in Drosophila. (A) In the absence of Hedgehog, the Patched receptor inhibits Smoothened probably by promoting the degradation or intracellular sequestration of Smoothened. The Ci protein is located in a protein complex and (more...)
Among the genes activated by Ci is the gene that encodes the Wnt protein Wingless, which helps pattern tissues in the fly embryo (discussed in Chapter 21). Another target gene is patched itself; the resulting increase in Patched protein on the cell surface inhibits further Hedgehog signaling--a form of negative feedback.
Many gaps in the Hedgehog signaling pathway still remain to be filled in. It is not known, for example, how Patched inhibits Smoothened, how Smoothened activates the pathway, how the proteolysis of Ci is regulated (although it is known that Ci phosphorylation by PKA is required for the processing), or how the release of the complex from microtubules and unprocessed Ci from the complex is controlled.
Even less is known about the Hedgehog pathway in vertebrate cells. In addition to there being at least three types of vertebrate Hedgehog proteins, there are two forms of Patched and three Ci-like proteins (Gli1, Gli2, and Gli3). Unlike in flies, Hedgehog signaling stimulates the transcription of the Gli genes, and it is unclear whether all of the Gli proteins undergo proteolytic processing, although there is evidence that Gli3 does. Inactivating mutations in one of the human patched genes, which leads to excessive Hedgehog signaling, occur frequently in the most common form of skin cancer (basal cell carcinoma), suggesting that Patched normally helps to keep skin cell proliferation in check.
The NF-κB proteins are latent gene regulatory proteins that lie at the heart of most inflammatory responses. These responses occur as a reaction to infection or injury and help protect the animal and its cells from these stresses. When excessive or inappropriate, however, inflammatory responses can also damage tissue and cause severe pain, as happens in joints in rheumatoid arthritis, for example. NF-κB proteins also have an important role in intercellular signaling during normal vertebrate development, although the extracellular signals that activate NF-κB in these circumstances are unknown. In Drosophila, however, genetic studies have identified both the extracellular and the intracellular proteins that activate the NF-κB family member Dorsal, which has a crucial role in specifying the dorsal-ventral axis of the developing fly embryo (discussed in Chapter 21). The same intracellular signaling pathway is also involved in defending the fly from infection, just as in vertebrates.
Two vertebrate cytokines are especially important in inducing inflammatory responses--tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1). Both are made by cells of the innate immune system, such as macrophages, in response to infection or tissue injury. These proinflammatory cytokines bind to cell-surface receptors and activate NF-κB, which is normally sequestered in an inactive form in the cytoplasm of almost all of our cells. Once activated, NF-κB turns on the transcription of more than 60 known genes that participate in inflammatory responses. Although TNF-α receptors and IL-1 receptors are structurally unrelated, they operate in much the same way.
There are five NF-κB proteins in mammals (RelA, RelB, c-Rel, NF-κB1, and NF-κB2), and they form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Inhibitory proteins calledIκB bind tightly to the dimers and hold them in an inactive state within large protein complexes in the cytoplasm. Signals such as TNF-α or IL-1 activate the dimers by triggering a signaling pathway that leads to the phosphorylation, ubiquitylation, and consequent degradation of IκB. The degradation of IκB exposes a nuclear localization signal on the NF-κB proteins, which now move into the nucleus and stimulate the transcription of specific genes. The phosphorylation of IκB is performed by a specific serine/threonine kinase called IκB kinase (IKK).
The mechanism by which the binding of a proinflammatory cytokine to its cell-surface receptors activates IκB kinase is illustrated for the TNF-α receptor in Figure 15-74. Ligand binding causes the cytosolic tails of the clustered receptors to recruit various adaptor proteins and cytoplasmic serine/threonine kinases. One of the recruited kinases is thought to be an IκB kinase kinase (IKKK) that directly phosphorylates and activates the IκB kinase (IKK).
The activation of NF-κB by TNF-α. Both TNF-α and its receptors are trimers. The binding of TNF-α causes a rearrangement of the clustered cytosolic tails of the receptors, which now recruit a number of intracellular signaling(more...)
Not all of the signaling proteins recruited to the cytosolic tail of the TNF-α receptor contribute to NF-κB activation, however. Some can trigger a MAP-kinase cascade, while others can activate a proteolytic cascade that leads toapoptosis (discussed in Chapter 17).
Thus far, we have discussed cell signaling mainly in animals, with a few diversions into yeasts and bacteria. But intercellular signaling is just as important for plants as it is for animals, although the mechanisms and molecules used are mainly different, as we discuss next.
Some signaling pathways that are especially important in animal development depend on proteolysis for at least part of their action. Notch receptors are activated by cleavage when Delta (or a related ligand) on another cell binds to them; the cleaved cytosolic tail of Notch migrates into the nucleus, where it stimulates gene transcription. In the Wntsignaling pathway, by contrast, the proteolysis of the latent gene regulatory protein β-catenin is inhibited when secreted Wnt proteins bind to their receptors; as a result, β-catenin accumulates in the nucleus and activates the transcription of Wnt target genes.
Hedgehog signaling in flies works much like Wnt signaling: in the absence of a signal, a bifunctional, cytoplasmicgene regulatory protein Ci is proteolytically cleaved to form a transcriptional repressor that keeps Hedgehog target genes silenced. The binding of Hedgehog to its receptor inhibits the proteolytic processing of Ci; as a result, the larger form of Ci accumulates in the nucleus and activates the transcription of Hedgehog-responsive genes. Signaling through the latent gene regulatory protein NF-κ B also depends on proteolysis. NF-κ B is normally held in an inactive state by the inhibitory protein Iκ B within a multiprotein complex in the cytoplasm. A variety of extracellular stimuli, including proinflammatory cytokines, trigger a phosphorylation cascade that ultimately phosphorylates Iκ B, marking it for degradation; this enables the freed NF-κ B to enter the nucleus and activate the transcription of its target genes.
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