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Cell_Biology_Alberts. In each case, the desensitization of the GPCRs depends on their phosphorylation by PKA, PKC, or a member of the family of GPCR kinases (GRKs), which includes the rhodopsin-specific kinase RK involved in rod photoreceptor desensitization discussed earlier. The GRKs phosphorylate multiple serines and threonines on a GPCR, but they do so only after ligand binding has activated the receptor, because it is the activated receptor that allosterically activates the GRK. 500 G-protein (transducin) molecules are activated 500 cyclic GMP phosphodiesterase molecules are activated 105 cyclic GMP molecules are hydrolyzed 106–107 Na+ ions per second are prevented from entering the cell for a period of ~1 second membrane potential is altered by 1 mV Figure 15–41 Amplification in the light-induced catalytic cascade in vertebrate rods. The red arrows indicate the steps where amplification occurs, with the thickness of the arrow roughly indicating the magnitude of the amplification.


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Cell_Biology_Alberts. Figure 15–42 The roles of GPCR kinases (GRKs) and arrestins in GPCR desensitization. A GRK phosphorylates only activated receptors because it is the activated GPCR that activates the GRK. The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein and also directs its endocytosis (not shown). Mice that are deficient in one form of arrestin fail to desensitize in response to morphine, for example, attesting to the importance of arrestins for desensitization. As with rhodopsin, once a receptor has been phosphorylated by a GRK, it binds with high affinity to a member of the arrestin family of proteins (Figure 15–42).


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Cell_Biology_Alberts. As with rhodopsin, once a receptor has been phosphorylated by a GRK, it binds with high affinity to a member of the arrestin family of proteins (Figure 15–42). The bound arrestin can contribute to the desensitization process in at least two ways. First, it prevents the activated receptor from interacting with G proteins. Second, it serves as an adaptor protein to help couple the receptor to the clathrin-dependent endocytosis machinery (discussed in Chapter 13), inducing receptor-mediated endocytosis. The fate of the internalized GPCR–arrestin complex depends on other proteins in the complex. In some cases, the receptor is dephosphorylated and recycled back to the plasma membrane for reuse. In others, it is ubiquitylated, endocytosed, and degraded in lysosomes (discussed later).


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Cell_Biology_Alberts. Receptor endocytosis does not necessarily stop the receptor from signaling. In some cases, the bound arrestin recruits other signaling proteins to relay the signal onward from the internalized GPCRs along new pathways.


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Cell_Biology_Alberts. GPCRs can indirectly activate or inactivate either plasma-membrane-bound enzymes or ion channels via G proteins. When an activated receptor stimulates a G protein, the G protein undergoes a conformational change that activates its α subunit, thereby triggering release of a βγ complex. Either component can then directly regulate the activity of target proteins in the plasma membrane. Some GPCRs either activate or inactivate adenylyl cyclase, thereby altering the intracellular concentration of the second messenger cyclic AMP. Others activate a phosphoinositide-specific phospholipase C (PLCβ), which generates two second messengers. One is inositol 1,4,5-trisphosphate (IP3), which releases Ca2+ from the ER and thereby increases the concentration of Ca2+ in the cytosol. The other is diacylglycerol, which remains in the plasma membrane and helps activate protein kinase C (PKC). An increase in cytosolic cyclic AMP or Ca2+ levels affects cells mainly by stimulating cAMP-dependent protein


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Cell_Biology_Alberts. which remains in the plasma membrane and helps activate protein kinase C (PKC). An increase in cytosolic cyclic AMP or Ca2+ levels affects cells mainly by stimulating cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinases (CaMkinases), respectively.


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Cell_Biology_Alberts. PKC, PKA, and CaM-kinases phosphorylate specific target proteins and thereby alter the activity of the proteins. Each type of cell has its own characteristic set of target proteins that is regulated in these ways, enabling the cell to make its own distinctive response to the second messengers. The intracellular signaling cascades activated by GPCRs greatly amplify the responses, so that many thousands of target protein molecules are changed for each molecule of extracellular signaling ligand bound to its receptor. The responses mediated by GPCRs are rapidly turned off when the extracellular signal is removed, and activated GPCRs are inactivated by phosphorylation and association with arrestins.


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Cell_Biology_Alberts. Like GPCRs, enzyme-coupled receptors are transmembrane proteins with their ligand-binding domain on the outer surface of the plasma membrane. Instead of having a cytosolic domain that associates with a trimeric G protein, however, their cytosolic domain either has intrinsic enzyme activity or associates directly with an enzyme. Whereas a GPCR has seven transmembrane segments, each subunit of an enzyme-coupled receptor typically has only one. GPCRs and enzyme-coupled receptors often activate some of the same signaling pathways. In this section, we describe some of the important features of signaling by enzyme-coupled receptors, with an emphasis on the most common class of these proteins, the receptor tyrosine kinases.


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Cell_Biology_Alberts. Many extracellular signal proteins act through receptor tyrosine kinases (RTKs). These include many secreted and cell-surface-bound proteins that control cell behavior in developing and adult animals. Some of these signal proteins and their RTKs are listed in Table 15–4. There are about 60 human RTKs, which can be classified into about 20 structural subfamilies, each dedicated to its complementary family of protein ligands. Figure 15–43 shows the basic structural features of a number of the families that operate in mammals. In all cases, the binding of the signal protein to the ligand-binding domain on the extracellular side of the receptor activates the tyrosine kinase domain on the cytosolic side. This leads to phosphorylation of tyrosine side chains on the cytosolic part of the receptor, creating phosphotyrosine docking sites for various intracellular signaling proteins that relay the signal.


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Cell_Biology_Alberts. How does the binding of an extracellular ligand activate the kinase domain on the other side of the plasma membrane? For a GPCR, ligand binding is thought to change the relative orientation of several of the transmembrane α helices, thereby shifting the position of the cytoplasmic loops relative to one another. It is unlikely, however, that a conformational change could propagate across the lipid bilayer EGF receptor SS SS SS insulin receptor, NGF receptor FGF receptor CYTOSOL cysteine-immunoglobulin-like domain Eph receptor kinase insert region fbronectin-type-III-like domain IGF1 PDGF VEGF receptor receptor, receptor MCSF receptor through a single transmembrane α helix. Instead, for most RTKs, ligand binding causes the receptors to dimerize, bringing the two cytoplasmic kinase domains together and thereby promoting their activation (Figure 15–44).


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Cell_Biology_Alberts. Dimerization stimulates kinase activity by a variety of mechanisms. In many cases, such as the insulin receptor, dimerization simply brings the kinase domains close to each other in an orientation that allows them to phosphorylate each other on specific tyrosines in the kinase active sites, thereby promoting conformational changes that fully activate both kinase domains. In other cases, such as the receptor for epidermal growth factor (EGF), the kinase is not activated by phosphorylation but by conformational changes brought about by interactions between the two kinase domains outside their active sites (Figure 15–45).


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Cell_Biology_Alberts. Figure 15–43 Some subfamilies of RTKs. Only one or two members of each subfamily are indicated. Note that in some cases, the tyrosine kinase domain is interrupted by a “kinase insert region” that is an extra segment emerging from the folded kinase domain. The functions of most of the cysteine-rich, immunoglobulinlike, and fibronectin-type-III-like domains are not known. Some of the ligands for the receptors shown are listed in Table 15–4, along with some representative responses that they mediate.


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Cell_Biology_Alberts. Figure 15–44 Activation of RTKs by dimerization. In the absence of extracellular signals, most RTKs exist as monomers in which the internal kinase domain is inactive. Binding of ligand brings two monomers together to form a dimer. In most cases, the close proximity in the dimer leads the two kinase domains to phosphorylate each other, which has two effects. First, phosphorylation at some tyrosines in the kinase domains promotes the complete activation of the domains. Second, phosphorylation at tyrosines in other parts of the receptors generates docking sites for intracellular signaling proteins, resulting in the formation of large signaling complexes that can then broadcast signals along multiple signaling pathways.


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Cell_Biology_Alberts. Mechanisms of dimerization vary widely among different RTK family members. In some cases, as shown here, the ligand itself is a dimer and brings two receptors together by binding them simultaneously. In other cases, a monomeric ligand can interact with two receptors simultaneously to bring them together, or two ligands can bind independently on two receptors to promote dimerization. In some RTKs—notably those in the insulin receptor family—the receptor is always a dimer (see Figure 15–43), and ligand binding causes a conformational change that brings the two internal kinase domains closer together. Although many RTKs are activated by transautophosphorylation as shown here, there are some important exceptions, including the EGF receptor illustrated in Figure 15–45. Phosphorylated Tyrosines on RTKs Serve as Docking Sites for Intracellular Signaling Proteins


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Cell_Biology_Alberts. Phosphorylated Tyrosines on RTKs Serve as Docking Sites for Intracellular Signaling Proteins Once the kinase domains of an RTK dimer are activated, they phosphorylate multiple additional sites in the cytosolic parts of the receptors, typically in disordered regions outside the kinase domain (see Figure 15–44). This phosphorylation creates high-affinity docking sites for intracellular signaling proteins. Each signaling protein binds to a particular phosphorylated site on the activated receptors because it contains a specific phosphotyrosine-binding domain that recognizes surrounding features of the polypeptide chain in addition to the phosphotyrosine.


Cell_Biology_Alberts

Cell_Biology_Alberts. Once bound to the activated RTK, a signaling protein may become phosphorylated on tyrosines and thereby activated. In many cases, however, the binding alone may be sufficient to activate the docked signaling protein, by either inducing a conformational change in the protein or simply bringing it near the protein that is next in the signaling pathway. Thus, receptor phosphorylation serves as a switch to trigger the assembly of an intracellular signaling complex, which can then relay the signal onward, often along multiple routes, to various destinations in the cell. Because different RTKs bind different combinations of these signaling proteins, they activate different responses.


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Cell_Biology_Alberts. Some RTKs use additional docking proteins to enlarge the signaling complex at activated receptors. Insulin and IGF1 receptor signaling, for example, depend on a specialized docking protein called insulin receptor substrate 1 (IRS1). IRS1 associates with phosphorylated tyrosines on the activated receptor and is then phosphorylated at multiple sites, thereby creating many more docking sites than could be accommodated on the receptor alone (see Figure 15–11). Proteins with SH2 Domains Bind to Phosphorylated Tyrosines


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Cell_Biology_Alberts. A whole menagerie of intracellular signaling proteins can bind to the phosphotyrosines on activated RTKs (or on docking proteins such as IRS1). They help to relay the signal onward, mainly through chains of protein–protein interactions mediated by modular interaction domains, as discussed earlier. Some of the docked proteins are enzymes, such as phospholipase C-γ (PLCγ), which functions in the same way as phospholipase C-β—activating the inositol phospholipid signaling pathway discussed earlier in connection with GPCRs (see Figures 15–28 and 15–29). Through this pathway, RTKs can increase cytosolic Ca2+ levels and activate PKC. Another enzyme that docks on these receptors is the cytoplasmic tyrosine kinase Src, which phosphorylates other signaling proteins on tyrosines. Yet another is phosphoinositide 3-kinase (PI 3-kinase), which phosphorylates lipids rather than proteins; as we discuss later, the phosphorylated lipids then serve as docking sites to attract various signaling proteins


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Cell_Biology_Alberts. 3-kinase (PI 3-kinase), which phosphorylates lipids rather than proteins; as we discuss later, the phosphorylated lipids then serve as docking sites to attract various signaling proteins to the plasma membrane.


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Cell_Biology_Alberts. The intracellular signaling proteins that bind to phosphotyrosines have varied structures and functions. However, they usually share highly conserved phosphotyrosine-binding domains. These can be either SH2 domains (for Src homology


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Cell_Biology_Alberts. Figure 15–45 Activation of the eGF receptor kinase. In the absence of ligand, the EGF receptor exists primarily as an inactive monomer. EGF binding results in a conformational change that promotes dimerization of the external domains. The receptor kinase domain, unlike that of many RTKs, is not activated by transautophosphorylation. Instead, dimerization orients the internal kinase domains into an asymmetric dimer, in which one kinase domain (the “activator”) pushes against the other kinase domain (the “receiver”), thereby causing an activating conformational change in the receiver. The active receiver domain then phosphorylates multiple tyrosines in the C-terminal tails of both receptors, generating docking sites for intracellular signaling proteins (see Figure 15–44).


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Cell_Biology_Alberts. region) or, less commonly, PTB domains (for phosphotyrosine-binding). By recognizing specific phosphorylated tyrosines, these small interaction domains enable the proteins that contain them to bind to activated RTKs, as well as to many other intracellular signaling proteins that have been transiently phosphorylated on tyrosines (Figure 15–46). As discussed previously, many signaling proteins also contain other interaction domains that allow them to interact specifically with other proteins as part of the signaling process. These domains include the SH3 domain, which binds to proline-rich motifs in intracellular proteins (see Figure 15–11).


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Cell_Biology_Alberts. Not all proteins that bind to activated RTKs via SH2 domains help to relay the signal onward. Some act to decrease the signaling process, providing negative feedback. One example is the c-Cbl protein, which can dock on some activated receptors and catalyze their ubiquitylation, covalently adding one or more ubiquitin molecules to specific sites on the receptor. This promotes the endocytosis and degradation of the receptors in lysosomes—an example of receptor down-regulation (see Figure 15–20). Endocytic proteins that contain ubiquitininteraction motifs (UIMs) recognize the ubiquitylated RTKs and direct them into clathrin-coated vesicles and, ultimately, into lysosomes (discussed in Chapter 13). Mutations that inactivate c-Cbl-dependent RTK down-regulation cause prolonged RTK signaling and thereby promote the development of cancer.


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Cell_Biology_Alberts. As is the case for GPCRs, ligand-induced endocytosis of RTKs does not always decrease signaling. In some cases, RTKs are endocytosed with their bound signaling proteins and continue to signal from endosomes or other intracellular compartments. This mechanism, for example, allows nerve growth factor (NGF) to bind to its specific RTK (called TrkA) at the end of a long nerve cell axon and signal to the cell body of the same cell a long distance away. Here, signaling endocytic Figure 15–46 The binding of SH2containing intracellular signaling proteins to an activated RTK.


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Cell_Biology_Alberts. (A) This drawing of a receptor for platelet-derived growth factor (PDGF) shows five phosphotyrosine docking sites, three in the kinase insert region and two on the C-terminal tail, to which the three signaling proteins shown bind as indicated. The numbers on the right indicate the positions of the tyrosines in the polypeptide chain. These binding sites have been identified by using recombinant DNA technology to mutate specific tyrosines in the receptor. Mutation of tyrosines 1009 and 1021, for example, prevents the binding and activation of PLCγ, so that receptor activation no longer stimulates the inositol phospholipid signaling pathway. The locations of the SH2 (red) and SH3 (blue) domains in the three signaling proteins are indicated. (Additional phosphotyrosine docking sites on this receptor are not shown, including those that bind the cytoplasmic tyrosine kinase Src and two adaptor proteins.) It is unclear how many signaling proteins can bind simultaneously to a single RTK. (B)


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Cell_Biology_Alberts. receptor are not shown, including those that bind the cytoplasmic tyrosine kinase Src and two adaptor proteins.) It is unclear how many signaling proteins can bind simultaneously to a single RTK. (B) The three-dimensional structure of an SH2 domain, as determined by x-ray crystallography. The binding pocket for phosphotyrosine is shaded in orange on the right, and a pocket for binding a specific amino acid side chain (isoleucine, in this case) is shaded in yellow on the left. The RTK polypeptide segment that binds the SH2 domain is shown in yellow (see also Figure 3–40). (C) The SH2 domain is a compact, “plug-in” module, which can be inserted almost anywhere in a protein without disturbing the protein’s folding or function (discussed in Chapter 3). Because each domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B,


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Cell_Biology_Alberts. for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B, based on data from G. Waksman et al., Cell 72:779–790, 1993. With permission from Elsevier. PDB code: 2SRC.) vesicles containing TrkA, with NGF bound on the lumenal side and signaling proteins docked on the cytosolic side, are transported along the axon to the cell body, where they signal the cell to survive.


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Cell_Biology_Alberts. Some signaling proteins are composed almost entirely of SH2 and SH3 domains and function as adaptors to couple tyrosine-phosphorylated proteins to other proteins that do not have their own SH2 domains (see Figure 15–11). Adaptor proteins of this type help to couple activated RTKs to the important signaling protein Ras, a monomeric GTPase that, in turn, can activate various downstream signaling pathways, as we now discuss. The GTPase Ras Mediates Signaling by Most RTKs The Ras superfamily consists of various families of monomeric GTPases, but only the Ras and Rho families relay signals from cell-surface receptors (Table 15–5). By interacting with different intracellular signaling proteins, a single Ras or Rho family member can coordinately spread the signal along several distinct downstream signaling pathways, thereby acting as a signaling hub.


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Cell_Biology_Alberts. There are three major, closely related Ras proteins in humans: H-, K-, and N-Ras (see Table 15–5). Although they have subtly different functions, they are thought to work in the same way, and we will refer to them simply as Ras. Like many monomeric GTPases, Ras contains one or more covalently attached lipid groups that help anchor the protein to the cytoplasmic face of the membrane, from where it relays signals to other parts of the cell. Ras is often required, for example, when RTKs signal to the nucleus to stimulate cell proliferation or differentiation, both of which require changes in gene expression. If Ras function is inhibited by various experimental approaches, the cell proliferation or differentiation responses normally induced by the activated RTKs do not occur. Conversely, 30% of human tumors express hyperactive mutant forms of Ras, which contribute to the uncontrolled proliferation of the cancer cells.


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Cell_Biology_Alberts. Like other GTP-binding proteins, Ras functions as a molecular switch, cycling between two distinct conformational states—active when GTP is bound and inactive when GDP is bound (Movie 15.7). As discussed earlier for monomeric GTPases in general, two classes of signaling proteins regulate Ras activity by influencing its transition between active and inactive states (see Figure 15–8). Ras guanine nucleotide exchange factors (Ras-GEFs) stimulate the dissociation of GDP and the subsequent uptake of GTP from the cytosol, thereby activating Ras. Ras GTPase-activating proteins (Ras-GAPs) increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating Ras. Hyperactive mutant forms of Ras are number of activated resistant to GAP-mediated GTPase stimulation and are locked permanently in the GTP-bound active state, which is why they promote the development of cancer.


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Cell_Biology_Alberts. GTP-bound active state, which is why they promote the development of cancer. But how do RTKs normally activate Ras? In principle, they could either activate a Ras-GEF or inhibit a Ras-GAP. Even though some GAPs bind directly (via their SH2 domains) to activated RTKs (see Figure 15–46A), it is the indirect coupling of the receptor to a Ras-GEF that drives Ras into its active state. The loss of function of a Ras-GEF has a similar effect to the loss of function of Ras itself. Activation of the other Ras superfamily proteins, including those of the Rho family, also occurs through the activation of GEFs. The particular GEF determines in which membrane the GTPase is activated and, by acting as a scaffold, it can also determine which downstream proteins the GTPase activates.


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Cell_Biology_Alberts. The GEF that mediates Ras activation by RTKs was discovered by genetic studies of eye development in Drosophila, where an RTK called Sevenless (Sev) is required for the formation of a photoreceptor cell called R7. Genetic screens for components of this signaling pathway led to the discovery of a Ras-GEF called Son-of-sevenless (Sos). Further genetic screens uncovered another protein, now called Grb2, which is an adaptor protein that links the Sev receptor to the Sos protein; the SH2 domain of the Grb2 adaptor binds to the activated receptor, while its two SH3 domains bind to Sos. Sos then promotes Ras activation. Biochemical and cell biological studies have shown that Grb2 and Sos also link activated RTKs to Ras in mammalian cells, revealing that this is a highly conserved mechanism in RTK signaling (Figure 15–47). Once activated, Ras activates various other signaling proteins to relay the signal downstream, as we discuss next.


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Cell_Biology_Alberts. Both the tyrosine phosphorylations and the activation of Ras triggered by activated RTKs are usually short-lived (Figure 15–48). Tyrosine-specific protein phosphatases quickly reverse the phosphorylations, and Ras-GAPs induce activated 0.1 0.05 Figure 15–47 How an RTK activates Ras. Grb2 recognizes a specific phosphorylated tyrosine on the activated receptor by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates the inactive Ras protein to replace its bound GDP by GTP, which activates Ras to relay the signal downstream.


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Cell_Biology_Alberts. Figure 15–48 Transient activation of Ras revealed by single-molecule fluorescence resonance energy transfer (FReT). (A) Schematic drawing of the experimental strategy. Cells of a human cancer cell line are genetically engineered to express a Ras protein that is covalently linked to yellow fluorescent protein (YFP). GTP that is labeled with a red fluorescent dye is microinjected into some of the cells. The cells are then stimulated with the extracellular signal protein EGF, and single fluorescent molecules of Ras-YFP at the inner surface of the plasma membrane are followed by video fluorescence microscopy in individual cells. When a fluorescent Ras-YFP molecule becomes activated, it exchanges unlabeled GDP for fluorescently labeled GTP; the energy emitted by the YFP now activates the fluorescent GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence


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Cell_Biology_Alberts. GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence from a previously yellow-green fluorescent spot at the plasma membrane. As shown in (B), activated Ras molecules can be detected after about 30 seconds of EGF stimulation. The red signal peaks at about 3 minutes and then decreases to baseline by 6 minutes. As Ras-GAP is found to be recruited to the same spots at the plasma membrane as Ras, it presumably plays a major part in rapidly shutting off the Ras signal. (Modified from H. Murakoshi et al., Proc. Natl Acad. Sci. USA 101:7317–7322, 2004. With permission from National Academy of Sciences.)


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Cell_Biology_Alberts. Ras to inactivate itself by hydrolyzing its bound GTP to GDP. To stimulate cells to proliferate or differentiate, these short-lived signaling events must be converted into longer-lasting ones that can sustain the signal and relay it downstream to the nucleus to alter the pattern of gene expression. One of the key mechanisms used for this purpose is a system of proteins called the mitogen-activated protein kinase module (MAP kinase module) (Figure 15–49). The three components of this system form a functional signaling module that has been remarkably well conserved during evolution and is used, with variations, in many different signaling contexts.


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Cell_Biology_Alberts. The three components are all protein kinases. The final kinase in the series is called simply MAP kinase (MAPK). The next one upstream from this is MAP kinase kinase (MAPKK): it phosphorylates and thereby activates MAP kinase. Next above that, receiving an activating signal directly from Ras, is MAP kinase kinase kinase (MAPKKK): it phosphorylates and thereby activates MAPKK. In the mammalian Ras–MAP-kinase signaling pathway, these three kinases are known by shorter names: Raf (= MAPKKK), Mek (= MAPKK), and Erk (=MAPK).


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Cell_Biology_Alberts. Once activated, the MAP kinase relays the signal downstream by phosphorylating various proteins in the cell, including transcription regulators and other protein kinases (see Figure 15–49). Erk, for example, enters the nucleus and phosphorylates one or more components of a transcription regulatory complex. This activates the transcription of a set of immediate early genes, so named because they turn on within minutes after an RTK receives an extracellular signal, even if protein synthesis is experimentally blocked with drugs. Some of these genes encode other transcription regulators that turn on other genes, a process that requires both protein synthesis and more time. In this way, the Ras–MAP-kinase signaling pathway conveys signals from the cell surface to the nucleus and alters the pattern of gene expression. Among the genes activated by this pathway are some that stimulate cell proliferation, such as the genes encoding G1 cyclins (discussed in Chapter 17).


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Cell_Biology_Alberts. Extracellular signals usually activate MAP kinases only transiently, and the period during which the kinase remains active influences the response. When EGF activates its receptors in a neural precursor cell line, for example, Erk MAP kinase activity peaks at 5 minutes and rapidly declines, and the cells later go on to divide. By contrast, when NGF activates its receptors on the same cells, Erk activity remains high for many hours, and the cells stop proliferating and differentiate into neurons. Many factors influence the duration and other features of the signaling response, including positive and negative feedback loops, which can combine to


Cell_Biology_Alberts

Cell_Biology_Alberts. Many factors influence the duration and other features of the signaling response, including positive and negative feedback loops, which can combine to Figure 15–49 The MAP kinase module activated by Ras. The three-component module begins with a MAP kinase kinase kinase called Raf. Ras recruits Raf to the plasma membrane and helps activate it. Raf then activates the MAP kinase kinase Mek, which then activates the MAP kinase Erk. Erk in turn phosphorylates a variety of downstream proteins, including other protein kinases, as well as transcription regulators in the nucleus. The resulting changes in protein activities and gene expression cause complex changes in cell behavior.


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Cell_Biology_Alberts. give responses that are either graded or switchlike and either brief or long lasting. In an example illustrated earlier, in Figure 15–19, MAP kinase activates a complex positive feedback loop to produce an all-or-none, irreversible response when frog oocytes are stimulated to mature by a brief exposure to the extracellular signal molecule progesterone. In many cells, MAP kinases activate a negative feedback loop by increasing the concentration of a protein phosphatase that removes the phosphate from MAP kinase. The increase in the phosphatase results from both an increase in the transcription of the phosphatase gene and the stabilization of the enzyme against degradation. In the Ras–MAP-kinase pathway shown in Figure 15–49, Erk also phosphorylates and inactivates Raf, providing another negative feedback loop that helps shut off the MAP kinase module.


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Cell_Biology_Alberts. Three-component MAP kinase signaling modules operate in all eukaryotic cells, with different modules mediating different responses in the same cell. In budding yeast, for example, one such module mediates the response to mating pheromone, another the response to starvation, and yet another the response to osmotic shock. Some of these MAP kinase modules use one or more of the same kinases and yet manage to activate different effector proteins and hence different responses. As discussed earlier, one way in which cells avoid cross-talk between the different parallel signaling pathways and ensure that each response is specific is to use scaffold proteins (see Figure 15–10A). In budding yeast cells, such scaffolds bind all or some of the kinases in each MAP kinase module to form a complex and thereby help to ensure response specificity (Figure 15–50).


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Cell_Biology_Alberts. Mammalian cells also use this scaffold strategy to prevent cross-talk between different MAP kinase modules. At least five parallel MAP kinase modules can operate in a mammalian cell. These modules make use of at least 12 MAP kinases, 7 MAPKKs, and 7 MAPKKKs. Two of these modules (terminating in MAP kinases called JNK and p38) are activated by different kinds of cell stresses, such as ultraviolet (UV) irradiation, heat shock, and osmotic stress, as well as by inflammatory cytokines; others mainly mediate responses to signals from other cells. Although the scaffold strategy provides precision and avoids cross-talk, it reduces the opportunities for amplification and spreading of the signal to different parts of the cell, which require at least some of the components to be diffusible. It is unclear to what extent the individual components of MAP kinase modules can dissociate from the scaffold during the activation process to permit amplification.


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Cell_Biology_Alberts. Figure 15–50 The organization of two MAP kinase modules by scaffold proteins in budding yeast. Budding yeast have at least six three-component MAP kinase modules involved in a variety of biological processes, including the two responses illustrated here—a mating response and the response to high osmolarity. (A) The mating response is triggered when a mating factor secreted by a yeast of opposite mating type binds to a GPCR. This activates a G protein, the βγ complex of which indirectly activates the MAPKKK (kinase A), which then relays the response onward. Once activated, the MAP kinase (kinase C) phosphorylates and thereby activates several proteins that mediate the mating response, in which the yeast cell stops dividing and prepares for fusion. The three kinases in this module are bound to scaffold protein 1. (B) In a second response, a yeast cell exposed to a high-osmolarity environment is induced to synthesize glycerol to increase its internal osmolarity. This response is mediated


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Cell_Biology_Alberts. to scaffold protein 1. (B) In a second response, a yeast cell exposed to a high-osmolarity environment is induced to synthesize glycerol to increase its internal osmolarity. This response is mediated by an osmolarity-sensing receptor protein and a different MAP kinase module bound to a second scaffold protein. (Note that the kinase domain of scaffold 2 provides the MAPKK activity of this module.) Although both pathways use the same MAPKKK (kinase A, green), there is no cross-talk between them because the kinases in each module are bound to different scaffold proteins, and the osmosensor is bound to the same scaffold protein as the particular kinase it activates.


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Cell_Biology_Alberts. Rho Family GTPases Functionally Couple Cell-Surface Receptors to the Cytoskeleton Besides the Ras proteins, the other class of Ras superfamily GTPases that relays signals from cell-surface receptors is the large Rho family (see Table 15–5). Rho family monomeric GTPases regulate both the actin and microtubule cytoskeletons, controlling cell shape, polarity, motility, and adhesion (discussed in Chapter 16); they also regulate cell-cycle progression, gene transcription, and membrane transport. They play a key part in the guidance of cell migration and nerve axon outgrowth, mediating cytoskeletal responses to the activation of a special class of guidance receptors. We focus on this aspect of Rho family function here.


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Cell_Biology_Alberts. The three best-characterized family members are Rho itself, Rac, and Cdc42, each of which affects multiple downstream target proteins. In the same way as for Ras, GEFs activate and GAPs inactivate the Rho family GTPases; there are more than 80 Rho-GEFs and more than 70 Rho-GAPs in humans. Some of the GEFs and GAPs are specific for one particular family member, whereas others are less specific. Unlike Ras, which is membrane-associated even when inactive (with GDP bound), inactive Rho family GTPases are often bound to guanine nucleotide dissociation inhibitors (GDIs) in the cytosol, which prevent the GTPases from interacting with their Rho-GEFs at the plasma membrane.


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Cell_Biology_Alberts. Signaling by extracellular signaling proteins of the ephrin family provides an example of how RTKs can activate a Rho GTPase. Ephrins bind and thereby activate members of the Eph family of RTKs (see Figure 15–43). One member of the Eph family is found on the surface of motor neurons and helps guide the migrating tip of the axon (called a growth cone) to its muscle target. The binding of a cell-surface ephrin protein activates the Eph receptor, causing the growth cones to collapse, thereby repelling them from inappropriate regions and keeping them on track. The response depends on a Rho-GEF called ephexin, which is stably associated with the cytosolic tail of the Eph receptor. When ephrin binding activates the Eph receptor, the receptor activates a cytoplasmic tyrosine kinase that phosphorylates ephexin on a tyrosine, enhancing the ability of ephexin to activate the Rho protein RhoA. The activated RhoA (RhoA-GTP) then regulates various downstream target proteins, including some


Cell_Biology_Alberts

Cell_Biology_Alberts. ephexin on a tyrosine, enhancing the ability of ephexin to activate the Rho protein RhoA. The activated RhoA (RhoA-GTP) then regulates various downstream target proteins, including some effector proteins that control the actin cytoskeleton, causing the growth cone to collapse (Figure 15–51).


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Cell_Biology_Alberts. Having considered how RTKs use GEFs and monomeric GTPases to relay signals into the cell, we now consider a second major strategy that RTKs use that depends on a quite different intracellular relay mechanism. Figure 15–51 Growth cone collapse mediated by Rho family GTPases. The binding of ephrin A1 proteins on an adjacent cell activates EphA4 RTKs on the growth cone. Phosphotyrosines on the activated Eph receptors recruit and activate a cytoplasmic tyrosine kinase to phosphorylate the receptor-associated Rho-GEF ephexin on a tyrosine. This enhances the ability of the ephexin to activate RhoA. RhoA then induces the growth cone to collapse by stimulating the myosin-dependent contraction of the actin cytoskeleton. PI 3-Kinase Produces Lipid Docking Sites in the Plasma Membrane


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Cell_Biology_Alberts. PI 3-Kinase Produces Lipid Docking Sites in the Plasma Membrane As mentioned earlier, one of the proteins that binds to the intracellular tail of RTK molecules is the plasma-membrane-bound enzyme phosphoinositide 3-kinase (PI 3-kinase). This kinase principally phosphorylates inositol phospholipids rather than proteins, and both RTKs and GPCRs can activate it. It plays a central part in promoting cell survival and growth.


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Cell_Biology_Alberts. Phosphatidylinositol (PI) is unique among membrane lipids because it can undergo reversible phosphorylation at multiple sites on its inositol head group to generate a variety of phosphorylated PI lipids called phosphoinositides. When activated, PI 3-kinase catalyzes phosphorylation at the 3 position of the inositol ring to generate several phosphoinositides (Figure 15–52). The production of PI(3,4,5)P3 matters most because it can serve as a docking site for various intracellular signaling proteins, which assemble into signaling complexes that relay the signal into the cell from the cytosolic face of the plasma membrane (see Figure 15–10C).


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Cell_Biology_Alberts. Notice the difference between this use of phosphoinositides and their use described earlier, in which PI(4,5)P2 is cleaved by PLCβ (in the case of GPCRs) or PLCγ(in the case of RTKs) to generate soluble IP3 and membrane-bound diacylglycerol (see Figures 15–28 and 15–29). By contrast, PI(3,4,5)P3 is not cleaved by either PLC. It is made from PI(4,5)P2 and then remains in the plasma membrane until specific phosphoinositide phosphatases dephosphorylate it. Prominent among these is the PTEN phosphatase, which dephosphorylates the 3 position of the inositol ring. Mutations in PTEN are found in many cancers: by prolonging signaling by PI 3-kinase, they promote uncontrolled cell growth.


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Cell_Biology_Alberts. There are various types of PI 3-kinases. Those activated by RTKs and GPCRs belong to class I. These are heterodimers composed of a common catalytic subunit and different regulatory subunits. RTKs activate class Ia PI 3-kinases, in which the regulatory subunit is an adaptor protein that binds to two phosphotyrosines on activated RTKs through its two SH2 domains (see Figure 15–46A). GPCRs activate class Ib PI 3-kinases, which have a regulatory subunit that binds to the βγ complex of an activated trimeric G protein (Gq) when GPCRs are activated by their extracellular ligand. The direct binding of activated Ras can also activate the common class I catalytic subunit. 6 1 2 3 4 5 diacylglycerol IP3 PLC CATALYZED BY PI 3-KINASE phosphatidylinositol (PI) PI 4-phosphate [PI(4)P] PI 3,4-bisphosphate [PI(3,4)P2] PI 4,5-bisphosphate [PI(4,5)P2] P P P P PP P P P P P P P P P


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Cell_Biology_Alberts. Figure 15–52 The generation of phosphoinositide docking sites by PI 3-kinase. PI 3-kinase phosphorylates the inositol ring on carbon atom 3 to generate the phosphoinositides shown at the bottom of the figure (diverting them away from the pathway leading to IP3 and diacylglycerol; see Figure 15–28). The most important phosphorylation (indicated in red) is of PI(4,5)P2 to PI(3,4,5)P3, which can serve as a docking site for signaling proteins with PI(3,4,5)P3-binding PH domains. Other inositol phospholipid kinases (not shown) catalyze the phosphorylations indicated by the green arrows. PI 3-phosphate PI 3,4,5-trisphosphate [PI(3)P] [PI(3,4,5)P3]


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Cell_Biology_Alberts. PI 3-phosphate PI 3,4,5-trisphosphate [PI(3)P] [PI(3,4,5)P3] Intracellular signaling proteins bind to PI(3,4,5)P3 produced by activated PI 3-kinase via a specific interaction domain, such as a pleckstrin homology (PH) domain, first identified in the platelet protein pleckstrin. PH domains function mainly as protein–protein interaction domains, and it is only a small subset of them that bind to PI(3,4,5)P3; at least some of these also recognize a specific membrane-bound protein as well as the PI(3,4,5)P3, which greatly increases the specificity of the binding and helps to explain why the signaling proteins with PI(3,4,5) P3-binding PH domains do not all dock at all PI(3,4,5)P3 sites. PH domains occur in about 200 human proteins, including the Ras-GEF Sos discussed earlier (see Figure 15–11).


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Cell_Biology_Alberts. One especially important PH-domain-containing protein is the serine/threonine protein kinase Akt. The PI-3-kinase–Akt signaling pathway is the major pathway activated by the hormone insulin. It also plays a key part in promoting the survival and growth of many cell types in both invertebrates and vertebrates, as we now discuss. The PI-3-Kinase–Akt Signaling Pathway Stimulates Animal Cells to Survive and Grow


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Cell_Biology_Alberts. The PI-3-Kinase–Akt Signaling Pathway Stimulates Animal Cells to Survive and Grow As discussed earlier, extracellular signals are usually required for animal cells to grow and divide, as well as to survive (see Figure 15–4). Members of the insulin-like growth factor (IGF) family of signal proteins, for example, stimulate many types of animal cells to survive and grow. They bind to specific RTKs (see Figure 15–43), which activate PI 3-kinase to produce PI(3,4,5)P3. The PI(3,4,5)P3 recruits two protein kinases to the plasma membrane via their PH domains—Akt (also called protein kinase B, or PKB) and phosphoinositide-dependent protein kinase 1 (PDK1), and this leads to the activation of Akt (Figure 15–53). Once activated,


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Cell_Biology_Alberts. PI(3,4,5)P3 PI(4,5)P2 PH domains CYTOSOL activated receptor tyrosine kinase activated PI 3-kinase PDK1 Akt phosphorylation and activation of Akt by PDK1 and mTOR dissociation active Akt Bad inactive apoptosis-inhibitory protein inactivated Bad INHIBITION OF APOPTOSIS PHOSPHORYLATION OF Bad 14-3-3 protein mTOR (in complex 2)P P P P P P P P P P P P P P P P P P


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Cell_Biology_Alberts. Figure 15–53 One way in which signaling through PI 3-kinase promotes cell survival. An extracellular survival signal activates an RTK, which recruits and activates PI 3-kinase. The PI 3-kinase produces PI(3,4,5)P3, which serves as a docking site for two serine/threonine kinases with PH domains—Akt and the phosphoinositide-dependent kinase PDK1—and brings them into proximity at the plasma membrane. The Akt is phosphorylated on a serine by a third kinase (usually mTOR in complex 2), which alters the conformation of the Akt so that it can be phosphorylated on a threonine by PDK1, which activates the Akt. The activated Akt now dissociates from the plasma membrane and phosphorylates various target proteins, including the Bad protein. When unphosphorylated, Bad holds one or more apoptosis-inhibitory proteins (of the Bcl2 family—discussed in Chapter 18) in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell


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Cell_Biology_Alberts. proteins (of the Bcl2 family—discussed in Chapter 18) in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell survival. As shown, the phosphorylated Bad binds to a ubiquitous cytosolic protein called 14-3-3, which keeps Bad out of action.


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Cell_Biology_Alberts. Akt phosphorylates various target proteins at the plasma membrane, as well as in the cytosol and nucleus. The effect on most of the known targets is to inactivate them; but the targets are such that these actions of Akt all conspire to enhance cell survival and growth, as illustrated for one cell survival pathway in Figure 15–53.


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Cell_Biology_Alberts. The control of cell growth by the PI-3-kinase–Akt pathway depends in part on a large protein kinase called TOR (named as the target of rapamycin, a bacterial toxin that inactivates the kinase and is used clinically as both an immunosuppressant and anticancer drug). TOR was originally identified in yeasts in genetic screens for rapamycin resistance; in mammalian cells, it is called mTOR, which exists in cells in two functionally distinct multiprotein complexes. mTOR complex 1 contains the protein raptor; this complex is sensitive to rapamycin, and it stimulates cell growth—both by promoting ribosome production and protein synthesis and by inhibiting protein degradation. Complex 1 also promotes both cell growth and cell survival by stimulating nutrient uptake and metabolism. mTOR complex 2 contains the protein rictor and is insensitive to rapamycin; it helps to activate Akt (see Figure 15–53), and it regulates the actin cytoskeleton via Rho family GTPases.


Cell_Biology_Alberts

Cell_Biology_Alberts. The mTOR in complex 1 integrates inputs from various sources, including extracellular signal proteins referred to as growth factors and nutrients such as amino acids, both of which help activate mTOR and promote cell growth. The growth factors activate mTOR mainly via the PI-3-kinase–Akt pathway. Akt activates mTOR in complex 1 indirectly by phosphorylating, and thereby inhibiting, a GAP called Tsc2. Tsc2 acts on a monomeric Ras-related GTPase called Rheb (see Table 15–5). Rheb in its active form (Rheb-GTP) activates mTOR in complex 1. The net result is that Akt activates mTOR and thereby promotes cell growth (Figure 15–54). We discuss how mTOR stimulates ribosome production and protein synthesis in Chapter 17 (see Figure 17–64).


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Cell_Biology_Alberts. As mentioned earlier, RTKs and GPCRs activate some of the same intracellular signaling pathways. Both, for example, can activate the inositol phospholipid pathway triggered by phospholipase C. Moreover, even when they activate different pathways, the different pathways can converge on the same target proteins. Figure 15–55 illustrates both of these types of signaling overlaps: it summarizes five parallel intracellular signaling pathways that we have discussed so far—one triggered by GPCRs, two triggered by RTKs, and two triggered by both kinds of receptors. Interactions among these pathways allow different extracellular signal molecules to modulate and coordinate each other’s effects. Figure 15–54 Activation of mTOR by the PI-3-kinase–Akt signaling pathway.


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Cell_Biology_Alberts. Figure 15–54 Activation of mTOR by the PI-3-kinase–Akt signaling pathway. (A) In the absence of extracellular growth factors, Tsc2 (a Rheb-GAP) keeps Rheb inactive; mTOR in complex 1 is inactive, and there is no cell growth. (B) In the presence of growth factors, activated Akt phosphorylates and inhibits Tsc2, thereby promoting the activation of Rheb. Activated Rheb (Rheb-GTP) helps activate mTOR in complex 1, which in turn stimulates cell growth. Figure 15–53 shows how growth factors (or survival signals) activate Akt. The Erk MAP kinase (see Figure 15–49) can also phosphorylate and inhibit Tsc2 and thereby activate mTOR. Thus, both the PI-3-kinase–Akt and Ras–MAP-kinase signaling pathways converge on mTOR in complex 1 to stimulate cell growth.


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Cell_Biology_Alberts. Tsc2 is short for tuberous sclerosis protein 2, and it is one component of a heterodimer composed of Tsc1 and Tsc2 (not shown); these proteins are so called because mutations in either gene encoding them cause the genetic disease tuberous sclerosis, which is associated with benign tumors that contain abnormally large cells. PI(3,4,5)P3 Some Enzyme-Coupled Receptors Associate with Cytoplasmic Tyrosine Kinases


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Cell_Biology_Alberts. Some Enzyme-Coupled Receptors Associate with Cytoplasmic Tyrosine Kinases Many cell-surface receptors depend on tyrosine phosphorylation for their activity and yet lack a tyrosine kinase domain. These receptors act through cytoplasmic tyrosine kinases, which are associated with the receptors and phosphorylate various target proteins, often including the receptors themselves, when the receptors bind their ligand. These tyrosine-kinase-associated receptors thus function in much the same way as RTKs, except that their kinase domain is encoded by a separate gene and is noncovalently associated with the receptor polypeptide chain. A variety of receptor classes belong in this category, including the receptors for antigen and interleukins on lymphocytes (discussed in Chapter 24), integrins (discussed in Chapter 19), and receptors for various cytokines and some hormones. As with RTKs, many of these receptors are either preformed dimers or are cross-linked into dimers by ligand binding.


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Cell_Biology_Alberts. Some of these receptors depend on members of the largest family of mammalian cytoplasmic tyrosine kinases, the Src family (see Figures 3–10 and 3–64), which includes Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk. These protein kinases all contain SH2 and SH3 domains and are located on the cytoplasmic side of the plasma membrane, held there partly by their interaction with transmembrane receptor proteins and partly by covalently attached lipid chains. Different family members are associated with different receptors and phosphorylate overlapping but distinct sets of target proteins. Lyn, Fyn, and Lck, for example, are each associated with different sets of receptors on lymphocytes. In each case, the kinase is activated when an extracellular ligand binds to the appropriate receptor protein. Src itself, as well as several other family members, can also bind to activated RTKs; in these cases, the receptor and cytoplasmic kinases mutually stimulate each other’s catalytic activity, thereby


Cell_Biology_Alberts

Cell_Biology_Alberts. Src itself, as well as several other family members, can also bind to activated RTKs; in these cases, the receptor and cytoplasmic kinases mutually stimulate each other’s catalytic activity, thereby strengthening and prolonging the signal (see Figure 15–51). There are even some G proteins (Gs and Gi) that can activate Src, which is one way that the activation of GPCRs can lead to tyrosine phosphorylation of intracellular signaling proteins and effector proteins.


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Cell_Biology_Alberts. Figure 15–55 Five parallel intracellular signaling pathways activated by GPCRs, RTKs, or both. In this simplified example, the five kinases (shaded yellow) at the end of each signaling pathway phosphorylate target proteins (shaded red), many of which are phosphorylated by more than one of the kinases. The phospholipase C activated by the two types of receptors is different: GPCRs activate PLCβ, whereas RTKs activate PLCγ (not shown). Although not shown, some GPCRs can also activate Ras, but they do so independently of Grb2, via a Ras-GEF that is activated by Ca2+ and diacylglycerol.


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Cell_Biology_Alberts. Another type of cytoplasmic tyrosine kinase associates with integrins, the main receptors that cells use to bind to the extracellular matrix (discussed in Chapter 19). The binding of matrix components to integrins activates intracellular signaling pathways that influence the behavior of the cell. When integrins cluster at sites of matrix contact, they help trigger the assembly of cell–matrix junctions called focal adhesions. Among the many proteins recruited into these junctions is the cytoplasmic tyrosine kinase called focal adhesion kinase (FAK), which binds to the cytosolic tail of one of the integrin subunits with the assistance of other proteins. The clustered FAK molecules phosphorylate each other, creating phosphotyrosine docking sites where the Src kinase can bind. Src and FAK then phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by RTKs. In this way, the two tyrosine kinases signal to the cell that it has


Cell_Biology_Alberts

Cell_Biology_Alberts. phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by RTKs. In this way, the two tyrosine kinases signal to the cell that it has adhered to a suitable substratum, where the cell can now survive, grow, divide, migrate, and so on.


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Cell_Biology_Alberts. The largest and most diverse class of receptors that rely on cytoplasmic tyrosine kinases to relay signals into the cell is the class of cytokine receptors, which we consider next. Cytokine Receptors Activate the JAK–STAT Signaling Pathway The large family of cytokine receptors includes receptors for many kinds of local mediators (collectively called cytokines), as well as receptors for some hormones, such as growth hormone and prolactin (Movie 15.8). These receptors are stably associated with cytoplasmic tyrosine kinases called Janus kinases (JAKs) (after the two-faced Roman god), which phosphorylate and activate transcription regulators called STATs (signal transducers and activators of transcription). STAT proteins are located in the cytosol and are referred to as latent transcription regulators because they migrate into the nucleus and regulate gene transcription only after they are activated.


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Cell_Biology_Alberts. Although many intracellular signaling pathways lead from cell-surface receptors to the nucleus, where they alter gene transcription (see Figure 15–55), the JAK–STAT signaling pathway provides one of the more direct routes. Cytokine receptors are dimers or trimers and are stably associated with one or two of the four known JAKs (JAK1, JAK2, JAK3, and Tyk2). Cytokine binding alters the arrangement so as to bring two JAKs into close proximity so that they phosphorylate each other, thereby increasing the activity of their tyrosine kinase domains. The JAKs then phosphorylate tyrosines on the cytoplasmic tails of cytokine receptors, creating phosphotyrosine docking sites for STATs (Figure 15–56). Some adaptor proteins can also bind to some of these sites and couple cytokine receptors to the Ras–MAP-kinase signaling pathway discussed earlier, but these will not be discussed here.


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Cell_Biology_Alberts. There are at least six STATs in mammals. Each has an SH2 domain that performs two functions. First, it mediates the binding of the STAT protein to a phosphotyrosine docking site on an activated cytokine receptor. Once bound, the JAKs phosphorylate the STAT on tyrosines, causing the STAT to dissociate from the receptor. Second, the SH2 domain on the released STAT now mediates its binding to a phosphotyrosine on another STAT molecule, forming either a STAT homodimer or a heterodimer. The STAT dimer then translocates to the nucleus, where, in combination with other transcription regulatory proteins, it binds to a specific cis-regulatory sequence in various genes and stimulates their transcription (see Figure 15–56). In response to the hormone prolactin, for example, which stimulates breast cells to produce milk, activated STAT5 stimulates the transcription of genes that encode milk proteins. Table 15–6 lists some of the more than 30 cytokines and hormones that activate the JAK–STAT


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Cell_Biology_Alberts. cells to produce milk, activated STAT5 stimulates the transcription of genes that encode milk proteins. Table 15–6 lists some of the more than 30 cytokines and hormones that activate the JAK–STAT pathway by binding to cytokine receptors.


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Cell_Biology_Alberts. Negative feedback regulates the responses mediated by the JAK–STAT pathway. In addition to activating genes that encode proteins mediating the cytokine-induced response, the STAT dimers can also activate genes that encode inhibitory proteins that help shut off the response. Some of these proteins bind to and inactivate phosphorylated JAKs and their associated phosphorylated receptors; others bind to phosphorylated STAT dimers and prevent them from binding to their DNA targets. Such negative feedback mechanisms, however, are not enough on their own to turn off the response. Inactivation of the activated JAKs and STATs requires dephosphorylation of their phosphotyrosines.


Cell_Biology_Alberts

Cell_Biology_Alberts. In all signaling pathways that use tyrosine phosphorylation, the tyrosine phosphorylations are reversed by protein tyrosine phosphatases. These phosphatases are as important in the signaling process as the protein tyrosine kinases that add the phosphates. Whereas only a few types of serine/threonine protein phosphatase Figure 15–56 The JAK–STAT signaling pathway activated by cytokines. The binding of the cytokine either causes two separate receptor polypeptide chains to dimerize (as shown) or re-orients the receptor chains in a preformed dimer. In either case, the associated JAKs are brought together so that they can phosphorylate each other on tyrosines to become fully activated, after which they phosphorylate the receptors to generate binding sites for the SH2 domains of STAT proteins. The JAKs also phosphorylate the STAT proteins, which dissociate from the receptor to form dimers and enter the nucleus to control gene expression.


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Cell_Biology_Alberts. catalytic subunits are responsible for removing phosphate groups from phosphorylated serines and threonines on proteins, there are about 100 protein tyrosine phosphatases encoded in the human genome, including some dual-specificity phosphatases that also dephosphorylate serines and threonines. Like tyrosine kinases, the tyrosine phosphatases occur in both cytoplasmic and transmembrane forms. Unlike serine/threonine protein phosphatases, which generally have broad specificity, most tyrosine phosphatases display exquisite specificity for their substrates, removing phosphate groups from only selected phosphotyrosines on a subset of proteins. Together, these phosphatases ensure that tyrosine phosphorylations are short-lived and that the level of tyrosine phosphorylation in resting cells is very low. They do not, however, simply continuously reverse the effects of protein tyrosine kinases; they are often regulated to act only at the appropriate time and place.


Cell_Biology_Alberts

Cell_Biology_Alberts. Having discussed the crucial role of tyrosine phosphorylation and dephosphorylation in the intracellular signaling pathways activated by many enzyme-coupled receptors, we now turn to a class of enzyme-coupled receptors that rely on serine and threonine phosphorylation. These receptor serine/threonine kinases activate an even more direct signaling pathway to the nucleus than does the JAK– STAT pathway. They directly phosphorylate latent transcription regulators called Smads, which then translocate into the nucleus to control gene transcription. Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and Smads


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Cell_Biology_Alberts. Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and Smads The transforming growth factor-β (TGFβ) superfamily consists of a large number (33 in humans) of structurally related, secreted, dimeric proteins. They act either as hormones or, more commonly, as local mediators to regulate a wide range of biological functions in all animals. During development, they regulate pattern formation and influence various cell behaviors, including proliferation, specification and differentiation, extracellular matrix production, and cell death. In adults, they are involved in tissue repair and in immune regulation, as well as in many other processes. The superfamily consists of the TGFβ/activin family and the larger bone morphogenetic protein (BMP) family.


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Cell_Biology_Alberts. All of these proteins act through enzyme-coupled receptors that are single-pass transmembrane proteins with a serine/threonine kinase domain on the cytosolic side of the plasma membrane. There are two classes of these receptor serine/threonine kinases—type I and type II—which are structurally similar homodimers. Each member of the TGFβ superfamily binds to a characteristic combination of type-I and type-II receptor dimers, bringing the kinase domains together so that the type-II receptor can phosphorylate and activate the type-I receptor, forming an active tetrameric receptor complex.


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Cell_Biology_Alberts. Once activated, the receptor complex uses a strategy for rapidly relaying the signal to the nucleus that is very similar to the JAK–STAT strategy used by cytokine receptors. The activated type-I receptor directly binds and phosphorylates a latent transcription regulator of the Smad family (named after the first two proteins identified, Sma in C. elegans and Mad in Drosophila). Activated TGFβ/ activin receptors phosphorylate Smad2 or Smad3, while activated BMP receptors phosphorylate Smad1, Smad5, or Smad8. Once one of these receptor-activated Smads (R-Smads) has been phosphorylated, it dissociates from the receptor and binds to Smad4 (called a co-Smad), which can form a complex with any of the five R-Smads. The Smad complex then translocates into the nucleus, where it associates with other transcription regulators and controls the transcription of specific target genes (Figure 15–57). Because the partner proteins in the nucleus vary depending on the cell type and state of the cell,


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Cell_Biology_Alberts. transcription regulators and controls the transcription of specific target genes (Figure 15–57). Because the partner proteins in the nucleus vary depending on the cell type and state of the cell, the genes affected vary.


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Cell_Biology_Alberts. Activated TGFβ receptors and their bound ligand are endocytosed by two distinct routes, one leading to further activation and the other leading to inactivation. The activation route depends on clathrin-coated vesicles and leads to early endosomes (discussed in Chapter 13), where most of the Smad activation occurs. An anchoring protein called SARA (for Smad anchor for receptor activation) has an important role in this pathway; it is concentrated in early endosomes and binds to both activated TGFβ receptors and Smads, increasing the efficiency of receptor-mediated Smad phosphorylation. The inactivation route depends on caveolae (discussed in Chapter 13) and leads to receptor ubiquitylation and degradation in proteasomes.


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Cell_Biology_Alberts. During the signaling response, the Smads shuttle continuously between the cytoplasm and the nucleus: they are dephosphorylated in the nucleus and exported to the cytoplasm, where they can be rephosphorylated by activated receptors. In this way, the effect exerted on the target genes reflects both the concentration of the extracellular signal and the time the signal continues to act on the cell-surface receptors (often several hours). Cells exposed to a morphogen at high concentration, or for a long time, or both, will switch on one set of genes, whereas cells receiving a lower or more transient exposure will switch on another set.


Cell_Biology_Alberts

Cell_Biology_Alberts. As in other signaling systems, negative feedback regulates the Smad pathway. Among the target genes activated by Smad complexes are those that encode inhibitory Smads, either Smad6 or Smad7. Smad7 (and possibly Smad6) binds to the cytosolic tail of the activated receptor and inhibits its signaling ability in at least three ways: (1) it competes with R-Smads for binding sites on the receptor, decreasing R-Smad phosphorylation; (2) it recruits a ubiquitin ligase called Smurf, which ubiquitylates the receptor, leading to receptor internalization and degradation (it is because Smurfs also ubiquitylate and promote the degradation of Smads that they are called Smad ubiquitylation regulatory factors, or Smurfs); and (3) it recruits a protein phosphatase that dephosphorylates and inactivates the receptor. In addition, the inhibitory Smads bind to the co-Smad, Smad4, and inhibit it, either by preventing its binding to R-Smads or by promoting its ubiquitylation and degradation.


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Cell_Biology_Alberts. Although receptor serine/threonine kinases operate mainly through the Smad pathway just described, they can also stimulate other intracellular signaling proteins such as MAP kinases and PI 3-kinase. Conversely, signaling proteins in other pathways can phosphorylate Smads and thereby influence signaling along the Smad pathway. There are various classes of enzyme-coupled receptors, the most common of which are receptor tyrosine kinases (RTKs), tyrosine-kinase-associated receptors, and receptor serine/threonine kinases. Figure 15–57 The Smad-dependent signaling pathway activated by TGFβ.


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Cell_Biology_Alberts. Figure 15–57 The Smad-dependent signaling pathway activated by TGFβ. The TGFβ dimer promotes the assembly of a tetrameric receptor complex containing two copies each of the type-I and type-II receptors. The type-II receptors phosphorylate specific sites on the type-I receptors, thereby activating their kinase domains and leading to phosphorylation of R-Smads such as Smad2 and Smad3. Smads open up to expose a dimerization surface when they are phosphorylated, leading to the formation of a trimeric Smad complex containing two R-Smads and the co-Smad, Smad4. The phosphorylated Smad complex enters the nucleus and collaborates with other transcription regulators to control the transcription of specific target genes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Ligand binding to RTKs causes their dimerization, which leads to activation of their kinase domains. These activated kinase domains phosphorylate multiple tyrosines on the receptors, producing a set of phosphotyrosines that serve as docking sites for a set of intracellular signaling proteins, which bind via their SH2 (or PTB) domains. One such signaling protein serves as an adaptor to couple some activated receptors to a Ras-GEF (Sos), which activates the monomeric GTPase Ras; Ras, in turn, activates a three-component MAP kinase signaling module, which relays the signal to the nucleus by phosphorylating transcription regulatory proteins. Another important signaling protein that can dock on activated RTKs is PI 3-kinase, which phosphorylates specific phosphoinositides to produce lipid docking sites in the plasma membrane for signaling proteins with phosphoinositide-binding PH domains, including the serine/threonine protein kinase Akt (PKB), which plays a key part in the control of cell


Cell_Biology_Alberts

Cell_Biology_Alberts. sites in the plasma membrane for signaling proteins with phosphoinositide-binding PH domains, including the serine/threonine protein kinase Akt (PKB), which plays a key part in the control of cell survival and cell growth. Many receptor classes, including some RTKs, activate Rho family monomeric GTPases, which functionally couple the receptors to the cytoskeleton.


Cell_Biology_Alberts

Cell_Biology_Alberts. Tyrosine-kinase-associated receptors depend on various cytoplasmic tyrosine kinases for their action. These kinases include members of the Src family, which associate with many kinds of receptors, and the focal adhesion kinase (FAK), which associates with integrins at focal adhesions. The cytoplasmic tyrosine kinases then phosphorylate a variety of signaling proteins to relay the signal onward. The largest family of receptors in this class is the cytokine receptor family. When stimulated by ligand binding, these receptors activate JAK cytoplasmic tyrosine kinases, which phosphorylate STATs. The STATs then dimerize, translocate to the nucleus, and activate the transcription of specific genes. Receptor serine/threonine kinases, which are activated by signal proteins of the TGFβsuperfamily, act similarly: they directly phosphorylate and activate Smads, which then oligomerize with another Smad, translocate to the nucleus, and regulate gene transcription.


Cell_Biology_Alberts

Cell_Biology_Alberts. Major changes in the behavior of a cell tend to depend on changes in the expression of numerous genes. Thus, many extracellular signaling molecules carry out their effects, in whole or in part, by initiating signaling pathways that change the activities of transcription regulators. There are numerous examples of gene regulation in both GPCR and enzyme-coupled receptor pathways (see Figures 15–27 and 15–49). In this section, we describe some of the less common signaling mechanisms by which gene expression can be controlled. We begin with several pathways that depend on regulated proteolysis to control the activity and location of latent transcription regulators. We then turn to a class of extracellular signal molecules that do not employ cell-surface receptors but enter the cell and interact directly with transcription regulators to perform their functions. Finally, we briefly discuss some of the mechanisms by which gene expression is controlled by the circadian rhythm: the daily cycle


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Cell_Biology_Alberts. directly with transcription regulators to perform their functions. Finally, we briefly discuss some of the mechanisms by which gene expression is controlled by the circadian rhythm: the daily cycle of light and dark.


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Cell_Biology_Alberts. The Receptor Notch Is a Latent Transcription Regulatory Protein


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Cell_Biology_Alberts. Signaling through the Notch receptor protein is used widely in animal development. As discussed in Chapter 22, it has a general role in controlling cell fate choices and regulating pattern formation during the development of most tissues, as well as in the continual renewal of tissues such as the lining of the gut. It is best known, however, for its role in the production of Drosophila neural cells, which usually arise as isolated single cells within an epithelial sheet of precursor cells. During this process, when a precursor cell commits to becoming a neural cell, it signals to its immediate neighbors not to do the same; the inhibited cells develop into epidermal cells instead. This process, called lateral inhibition, depends on a contact-dependent signaling mechanism that is activated by a single-pass trans-membrane signal protein called Delta, displayed on the surface of the future neural cell. By binding to the Notch receptor protein on a neighboring cell, Delta signals to the


Cell_Biology_Alberts

Cell_Biology_Alberts. by a single-pass trans-membrane signal protein called Delta, displayed on the surface of the future neural cell. By binding to the Notch receptor protein on a neighboring cell, Delta signals to the neighbor not to become neural (Figure 15–58). When this signaling process is defective, a huge excess of neural cells is produced at the expense of epidermal cells, which is lethal.


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Cell_Biology_Alberts. Notch is a single-pass transmembrane protein that requires proteolytic processing to function. It acts as a latent transcription regulator and provides the simplest and most direct signaling pathway known from a cell-surface receptor to the nucleus. When activated by the binding of Delta on another cell, a plasma-membrane-bound protease cleaves off the cytoplasmic tail of Notch, and the released tail translocates into the nucleus to activate the transcription of a set of Notch-response genes. The Notch tail fragment acts by binding to a DNA-binding protein, converting it from a transcriptional repressor into a transcriptional activator.


Cell_Biology_Alberts

Cell_Biology_Alberts. The Notch receptor undergoes three successive proteolytic cleavage steps, but only the last two depend on Delta binding. As part of its normal biosynthesis, it is cleaved in the Golgi apparatus to form 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 an extracellular protease. A final cleavage quickly follows, cutting free the cytoplasmic tail of the activated receptor (Figure 15–59). Note that, unlike most receptors, the activation of Notch is irreversible; once activated by ligand binding, the protein cannot be used again.


Cell_Biology_Alberts

Cell_Biology_Alberts. This final cleavage of the Notch tail occurs just within the transmembrane segment, and it is mediated by a protease complex called γ-secretase, which is also responsible for the intramembrane cleavage of various other proteins. One of its essential subunits is Presenilin, so called because mutations in the gene encoding it are a frequent cause of early-onset, familial Alzheimer’s disease, a form of presenile dementia. The protease complex is thought to contribute to this and other forms of Alzheimer’s disease by generating extracellular peptide fragments from a transmembrane neuronal protein; the fragments accumulate in excessive amounts and form aggregates of misfolded protein called amyloid plaques, which may injure nerve cells and contribute to their degeneration and loss.


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Cell_Biology_Alberts. Both Notch and Delta are glycoproteins, and their interaction is regulated by the glycosylation of Notch. The Fringe family of glycosyl transferases, in particular, 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 Bind to Frizzled Receptors and Inhibit the Degradation of β-Catenin Wnt proteins are secreted signal molecules that act as local mediators and morphogens 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 as a morphogen


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Cell_Biology_Alberts. Figure 15–58 lateral inhibition mediated by Notch and Delta during neural 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 signaling is mediated by the ligand Delta, which appears on the surface of the future neural cell and binds to Notch receptor proteins on the neighboring cells. In many tissues, all the cells in a cluster initially express both Delta and Notch, and a competition occurs, with one cell emerging as winner, expressing Delta strongly and inhibiting its neighbors from doing likewise. In other cases, additional factors interact with Delta or Notch to make some cells susceptible to the lateral inhibition signal and others unresponsive to it.


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Cell_Biology_Alberts. CYTOSOL 2 3 CYTOSOL Delta signaling cell plasma membrane target cell plasma membrane 9 EGF-like domains TRANSPORT TO PLASMA MEMBRANE BINDING TO DELTA, ENDOCYTOSIS OF DELTA– NOTCH-FRAGMENT COMPLEX, AND CLEAVAGE AT SITE 2 CLEAVAGE AT SITE 3 NOTCH TAIL MIGRATES TO NUCLEUS EXTRACELLULAR SPACE ENDOCYTOSIS


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Cell_Biology_Alberts. Figure 15–59 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 receptor that is then displayed on the cell surface. The binding to Delta on a neighboring cell triggers the next two proteolytic steps: the complex of Delta and the Notch fragment to which it is bound is endocytosed by the Delta-expressing cell, exposing the extracellular cleavage site in the transmembrane Notch subunit. Note that Notch and Delta interact through their repeated EGF-like domains. The released Notch tail migrates into the nucleus, where it binds to the Rbpsuh protein, which it converts from a transcriptional repressor to a transcriptional activator.


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Cell_Biology_Alberts. in wing development, while in mice, the Int1 gene was found because it promoted the development of breast tumors when activated by the integration of a virus next to it. Both of these genes encode Wnt proteins. Wnts are unusual as secreted proteins in that they have a fatty acid chain covalently attached to their N-terminus, which increases their binding to cell surfaces. There are 19 Wnts in humans, each having distinct, but often overlapping, functions.


Cell_Biology_Alberts

Cell_Biology_Alberts. Wnts can activate at least two types of intracellular signaling pathways. Our primary focus here is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is centered on the latent transcription regulator β-catenin. A second pathway, called the planar polarity pathway, coordinates the polarization of cells in the plane of a developing epithelium and depends on Rho family GTPases. Both of these pathways begin with the binding of Wnts to Frizzled family cell-surface receptors, which are seven-pass transmembrane proteins that resemble GPCRs in structure but do not generally work through the activation of G proteins. Instead, when activated by Wnt binding, Frizzled proteins recruit the scaffold protein Dishevelled, which helps relay the signal to other signaling molecules.


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Cell_Biology_Alberts. The Wnt/β-catenin pathway acts by regulating the proteolysis of the multi-functional protein β-catenin (or Armadillo in flies). A portion of the cell’s β-catenin is located at cell–cell junctions and thereby contributes to the control of cell– cell adhesion (discussed in Chapter 19), while the remaining β-catenin is rapidly degraded in the cytoplasm. Degradation depends on a large protein degradation complex, which binds β-catenin and keeps it out of the nucleus while promoting its degradation. The complex contains at least four other proteins: a protein kinase called casein kinase 1 (CK1) phosphorylates the β-catenin on a serine, priming it for further phosphorylation by another protein kinase called glycogen synthase kinase 3 (GSK3); this final phosphorylation marks the protein for ubiquitylation and rapid degradation in proteasomes. Two scaffold proteins called axin and Adenomatous polyposis coli (APC) hold the protein complex together (Figure 15–60A). APC gets its name from the


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Cell_Biology_Alberts. and rapid degradation in proteasomes. Two scaffold proteins called axin and Adenomatous polyposis coli (APC) hold the protein complex together (Figure 15–60A). APC gets its name from the finding that 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 and can eventually become malignant. (This APC should not be confused with the anaphase-promoting complex, or APC/C, that plays a central part in selective protein degradation during the cell cycle—see Figure 17–15A.)


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Cell_Biology_Alberts. Wnt proteins regulate β-catenin proteolysis by binding to both a Frizzled protein and a co-receptor that is related to the low-density lipoprotein (LDL) receptor (discussed in Chapter 13) and is therefore called an LDL-receptor-related protein (LRP). In a poorly understood process, the activated receptor complex recruits the Dishevelled scaffold and promotes the phosphorylation of the LRP receptor by the two protein kinases, GSK3 and CK1. Axin is brought to the receptor complex and inactivated, thereby disrupting the β-catenin degradation complex in the cytoplasm. In this way, the phosphorylation and degradation of β-catenin are prevented, enabling unphosphorylated β-catenin to accumulate and translocate to the nucleus, where it alters the pattern of gene transcription (Figure 15–60B).


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Cell_Biology_Alberts. Figure 15–60 The Wnt/β-catenin signaling pathway. (A) In the absence of a Wnt signal, β-catenin that is not bound to cell–cell adherens junctions (not shown) interacts with a degradation complex containing APC, axin, GSK3, and CK1. In this complex, β-catenin is phosphorylated by CK1 and then by GSK3, triggering its ubiquitylation and degradation in proteasomes. Wnt-responsive genes are kept inactive by the Groucho co-repressor protein bound to the transcription regulator LEF1/TCF. (B) Wnt binding to Frizzled and LRP clusters the two co-receptors together, and the cytosolic tail of LRP is phosphorylated by GSK3 and then by CK1. Axin binds to the phosphorylated LRP and is inactivated and/or degraded, resulting in disassembly of the degradation complex. The phosphorylation of β-catenin is thereby prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/ TCF, displaces the co-repressor Groucho, and acts as a coactivator to stimulate the


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Cell_Biology_Alberts. prevented, and unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to LEF1/ TCF, displaces the co-repressor Groucho, and acts as a coactivator to stimulate the transcription of Wnt target genes. The scaffold protein Dishevelled is required for the signaling pathway to operate; it binds to Frizzled and becomes phosphorylated (not shown), but its precise role is unknown.


Cell_Biology_Alberts

Cell_Biology_Alberts. In the absence of Wnt signaling, Wnt-responsive genes are kept silent by an inhibitory complex of transcription regulatory proteins. The complex includes proteins of the LEF1/TCF family bound to a co-repressor protein of the Groucho family (see Figure 15–60A). In response to a Wnt signal, β-catenin enters the nucleus and binds to the LEF1/TCF proteins, displacing Groucho. The β-catenin now functions as a coactivator, inducing the transcription of the Wnt target genes (see Figure 15–60B). Thus, as in the case of Notch signaling, Wnt/β-catenin signaling triggers a switch from transcriptional repression to transcriptional activation.


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Cell_Biology_Alberts. Among the genes activated by β-catenin is Myc, which encodes a protein (Myc) that is an important regulator of cell growth and proliferation (discussed in Chapter 17). Mutations of the Apc gene occur in 80% of human colon cancers (discussed in Chapter 20). These mutations inhibit the protein’s ability to bind β-catenin, so that β-catenin accumulates in the nucleus and stimulates the transcription of c-Myc and other Wnt target genes, even in the absence of Wnt signaling. The resulting uncontrolled cell growth and proliferation promote the development of cancer. Various secreted inhibitory proteins regulate Wnt signaling in development. Some bind to the LRP receptors and promote their down-regulation, whereas others compete with Frizzled receptors for secreted Wnts. In Drosophila at least, Wnts activate negative feedback loops, in which Wnt target genes encode proteins that help shut the response off; some of these proteins inhibit Dishevelled, and others are secreted inhibitors.


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Cell_Biology_Alberts. Hedgehog Proteins Bind to Patched, Relieving Its Inhibition of Smoothened Hedgehog proteins and Wnt proteins act in similar ways. Both are secreted signal molecules, which act as local mediators and morphogens in many developing invertebrate and vertebrate tissues. Both proteins are modified by covalently attached lipids, depend on secreted or cell-surface-bound heparan sulfate proteoglycans (discussed in Chapter 19) for their action, and activate latent transcription regulators by inhibiting their degradation. They both trigger a switch from transcriptional repression to transcriptional activation, and excessive signaling along either pathway in adult cells can lead to cancer. They even use some of the same intracellular signaling proteins and sometimes collaborate to mediate a response.


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Cell_Biology_Alberts. The Hedgehog proteins were discovered in Drosophila, where this protein family has only one member. Mutation of the Hedgehog gene produces a larva covered with spiky processes (denticles), like a hedgehog. At least three genes encode Hedgehog proteins in vertebrates—Sonic, Desert, and Indian hedgehog. The active forms of all Hedgehog proteins are covalently coupled to cholesterol, as well as to a fatty acid chain. The cholesterol is added during an unusual processing step, in which a precursor protein cleaves itself to produce a smaller, cholesterol-containing signal protein. Most of what we know about the Hedgehog signaling pathway came initially from genetic studies in flies, and it is the fly pathway that we summarize here.


Cell_Biology_Alberts

Cell_Biology_Alberts. The effects of Hedgehog are mediated by a latent transcription regulator called Cubitus interruptus (Ci), the regulation of which is reminiscent of the regulation of β-catenin by Wnts. In the absence of a Hedgehog signal, Ci is ubiquitylated and proteolytically cleaved in proteasomes. Instead of being completely degraded, however, Ci is processed to form a smaller fragment, which accumulates in the nucleus, where it acts as a transcriptional repressor, helping to keep Hedgehog-responsive genes silent. The proteolytic processing of the Ci protein depends on its phosphorylation by three protein kinases—PKA and two kinases also used in the Wnt pathway, namely GSK3 and CK1. As in the Wnt pathway, the proteolytic processing occurs in a multiprotein complex. The complex includes the protein kinase Fused and a scaffold protein Costal2, which stably associates with Ci, recruits the three other kinases, and binds the complex to microtubules, thereby keeping unprocessed Ci out of the nucleus


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Cell_Biology_Alberts. Fused and a scaffold protein Costal2, which stably associates with Ci, recruits the three other kinases, and binds the complex to microtubules, thereby keeping unprocessed Ci out of the nucleus (Figure 15–61A).


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Cell_Biology_Alberts. Patched inactive Smoothened microtubule Costal2 Fused large Ci protein PHOSPHORYLATION OF Ci BY PKA, GSK3, AND CK1 iHog Ig-like domain fbronectin-type-III-like domain vesicle cleaved Ci protein in complex with co-repressor Hedgehog functions by blocking the proteolytic processing of Ci, thereby changing it into a transcriptional activator. It does this by a convoluted signaling process that depends on three transmembrane proteins: Patched, iHog, and Smoothened. Patched is predicted to cross the plasma membrane 12 times, and, although much of it is in intracellular vesicles, some is on the cell surface where it can bind the Hedgehog protein. iHog is also on the cell surface and is thought to serve as a co-receptor for Hedgehog. Smoothened is a seven-pass transmembrane protein with a structure very similar to a GPCR, but it does not seem to act as a Hedgehog receptor or even as an activator of G proteins; it is controlled by Patched and iHog.


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Cell_Biology_Alberts. In the absence of a Hedgehog signal, Patched employs an unknown mechanism to keep Smoothened sequestered and inactive in intracellular vesicles (see Figure 15–61A). The binding of Hedgehog to iHog and Patched inhibits the activity of Patched and induces its endocytosis and degradation. The result is that Smoothened is liberated from inhibition and translocates to the plasma membrane, where it recruits the protein complex containing Ci, Fused, and Costal2.


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Cell_Biology_Alberts. Figure 15–61 Hedgehog signaling in Drosophila. (A) In the absence of Hedgehog, most Patched is in intracellular vesicles (not shown), where it keeps Smoothened inactive and sequestered. The Ci protein is bound in a cytosolic protein degradation complex, which includes the protein kinase Fused and the scaffold protein Costal2. Costal2 recruits three other protein kinases (PKA, GSK3, and CK1; not shown), which phosphorylate Ci. Phosphorylated Ci is ubiquitylated and then cleaved in proteasomes (not shown) to form a transcriptional repressor, which accumulates in the nucleus to help keep Hedgehog target genes inactive.


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Cell_Biology_Alberts. (B) Hedgehog binding to iHog and Patched removes the inhibition of Smoothened by Patched. Smoothened is phosphorylated by PKA and CK1 and translocates to the plasma membrane, where it recruits the complex containing Fused, Costal2, and Ci. Costal2 releases unprocessed Ci, which accumulates in the nucleus and activates the transcription of Hedgehog target genes. Many details in the pathway are poorly understood, including the role of Fused. Costal2 is no longer able to bind the other three kinases, and so Ci is no longer cleaved and can now enter the nucleus and activate the transcription of Hedgehog target genes (Figure 15–61B). Among the genes activated by Ci is Patched itself; the resulting increase in Patched protein on the cell surface inhibits further Hedgehog signaling—providing another example of negative feedback.


Cell_Biology_Alberts

Cell_Biology_Alberts. Many gaps remain in our understanding of the Hedgehog signaling pathway. It is not known, for example, how Patched keeps Smoothened inactive and intracellular. As the structure of Patched resembles a transmembrane transporter protein, it has been proposed that it may transport a small molecule into the cell that keeps Smoothened sequestered in vesicles.


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Cell_Biology_Alberts. Even less is known about the more complex Hedgehog pathway in vertebrate cells. In addition to there being at least three types of vertebrate Hedgehog proteins, there are three Ci-like transcription regulator proteins (Gli1, Gli2, and Gli3) downstream of Smoothened. Gli2 and Gli3 are most similar to Ci in structure and function, and Gli3 has been shown to undergo proteolytic processing like Ci and to act as either a transcriptional repressor or a transcriptional activator. Moreover, in vertebrates, Smoothened, upon activation, becomes localized to the surface of the primary cilium (discussed in Chapter 16), where the Gli proteins are also concentrated, thereby increasing the speed and efficiency of signaling.


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Cell_Biology_Alberts. Hedgehog signaling can promote cell proliferation, and excessive Hedgehog signaling can lead to cancer. Inactivating mutations in one of the two human Patched genes, for example, which lead to excessive Hedgehog signaling, occur frequently in basal cell carcinoma of the skin, the most common form of cancer in Caucasians. A small molecule called cyclopamine, made by a meadow lily, is being used to treat cancers associated with excessive Hedgehog signaling. It blocks Hedgehog signaling by binding tightly to Smoothened and inhibiting its activity. It was originally identified because it causes severe developmental defects in the progeny of sheep grazing on such lilies; these include the presence of a single central eye (a condition called cyclopia), which is also seen in mice that are deficient in Hedgehog signaling.


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Cell_Biology_Alberts. The NFκB proteins are latent transcription regulators that are present in most animal cells and are central to many stressful, inflammatory, and innate immune responses. These responses occur as a reaction to infection or injury and help protect stressed multicellular organisms and their cells (discussed in Chapter 24). An excessive or inappropriate inflammatory response in animals can also damage tissue and cause severe pain, and chronic inflammation can lead to cancer; as in the case of Wnt and Hedgehog signaling, excessive NFκB signaling is found in a number of human cancers. NFκB proteins also have important roles during normal animal development: the Drosophila NFκB family member Dorsal, for example, has a crucial role in specifying the dorsal–ventral axis of the developing fly embryo (discussed in Chapter 22).


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Cell_Biology_Alberts. Various cell-surface receptors activate the NFκB signaling pathway in animal cells. Toll receptors in Drosophila and Toll-like receptors in vertebrates, for example, recognize pathogens and activate this pathway in triggering innate immune responses (discussed in Chapter 24). The receptors for tumor necrosis factor α (TNFα) and interleukin-1 (IL1), which are vertebrate cytokines especially important in inducing inflammatory responses, also activate this signaling pathway. The Toll, Toll-like, and IL1 receptors belong to the same family of proteins, whereas TNF receptors belong to a different family; all of them, however, act in similar ways to activate NFκB. When activated, they trigger a multiprotein ubiquitylation and phosphorylation cascade that releases NFκB from an inhibitory protein complex, so that it can translocate to the nucleus and turn on the transcription of hundreds of genes that participate in inflammatory and innate immune responses.


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Cell_Biology_Alberts. There are five NFκB proteins in mammals (RelA, RelB, c-Rel, NFκB1, and NFκB2), and they form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Inhibitory proteins called IκB bind tightly to the dimers and hold them in an inactive state within the cytoplasm of unstimulated cells. There are three major IκB proteins in mammals (IκB α, β, and ε), and the signals that release NFκB dimers do so by triggering a signaling pathway that leads to the phosphorylation, ubiquitylation, and consequent degradation of the IκB proteins (Figure 15–62).


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Cell_Biology_Alberts. Among the genes activated by the released NFκB is the gene that encodes IκBα. This activation leads to increased synthesis of IκBα protein, which binds to NFκB and inactivates it, creating a negative feedback loop (Figure 15–63A). Experiments on TNFα-induced responses, as well as computer modeling studies of the responses, indicate that the negative feedback produces two types of NFκB responses, depending on the duration of the TNFα stimulus; importantly, the two types of responses induce different patterns of gene expression (Figure 15–63B, C, and D). The negative feedback through IκBα is required for both types of responses: in cells deficient in IκBα, even a short exposure to TNFαinduces a sustained activation of NFκB, without oscillations, and all of the NFκB-responsive genes are activated.


Cell_Biology_Alberts

Cell_Biology_Alberts. Thus far, we have focused on the mechanisms by which extracellular signal molecules use cell-surface receptors to initiate changes in gene expression. We now turn to a class of extracellular signals that bypasses the plasma membrane entirely and controls, in the most direct way possible, transcription regulatory proteins inside the cell. Various small, hydrophobic signal molecules diffuse directly across the plasma membrane of target cells and bind to intracellular receptors that are transcription regulators. These signal molecules include steroid hormones, thyroid hormones, retinoids, and vitamin D. Although they differ greatly from one another in both


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Cell_Biology_Alberts. Figure 15–62 The activation of the NFκb pathway by TNFα. Both TNFα and its receptors are trimers. The binding of TNFα causes a rearrangement of the clustered cytosolic tails of the receptors, which now recruit various signaling proteins, resulting in the activation of a protein kinase that phosphorylates and activates IκB kinase kinase (IKK). IKK is a heterotrimer composed of two kinase subunits (IKKα and IKKβ) and a regulatory subunit called NEMO. IKKβ then phosphorylates IκB on two serines, which marks the protein for ubiquitylation and degradation in proteasomes. The released NFκB translocates into the nucleus, where, in collaboration with coactivator proteins, it stimulates the transcription of its target genes. gene A on, gene B off gene A on, gene B on (A) (B) (C) KEY amount of nuclear NF˜B gene A expression gene B expression