+ Wild-type gene that restores interferon responsiveness

No Yes

No Yes

Growth in HAT

Killed by expressed medium 6-thioguanine

No Yes

No Yes

No Yes

No Yes


+ Interferon

+ Interferon

▲ EXPERIMENTAL FIGURE 14-10 Mutagenized cells carrying an interferon-responsive reporter gene were used to identify JAKs and STATs as essential signal-transduction proteins. A reporter gene was constructed consisting of an interferon-responsive promoter upstream of the bacterial gene encoding GPRT, a key enzyme in the purine salvage pathway (see Figure 6-39). (a) Introduction of this construct into mammalian cells lacking the mammalian homolog HGPRT yielded reporter cells that grew in HAT medium and were killed by 6-thioguanine in the presence but not the absence of interferon. (b) Following treatment of reporter cells with a mutagen, cells with defects In the signaling pathway Initiated by Interferon do not Induce GPRT In response to interferon and thus cannot incorporate the toxic purine 6-thioguanine. Restoration of Interferon responsiveness by functional complementation with wild-type DNA clones Identified genes encoding JAKs and STATs. See the text for details. [See R. McKendry et al., 1991, Proc. Nat'l. Acad. Sci. USA 88:11455; D. Watling et al., 1993, Nature 366:166; and G. Stark and A. Gudkov, 1999, Human Mol. Genet. 8:1925.]

in the culture medium into ribonucleotides and then into DNA or RNA. As shown in Figure 14-10a, HGPRT-negative cells carrying the reporter gene responded to interferon treatment by expressing GPRT and thus acquiring the ability to grow in HAT medium. This medium does not allow growth of cells lacking GPRT or HGPRT, since synthesis of purines by the cells is blocked by aminopterin (the A in HAT), and thus DNA synthesis is dependent on incorporation of purines from the culture medium (see Figure 6-39). Simultaneously the cells acquired sensitivity to killing by the purine analog 6-thioguanine, which is converted into the corresponding ribonucleotide by GPRT; incorporation of this purine into DNA in place of guanosine eventually causes cell death.

The reporter cells were then heavily treated with mutagens in an attempt to inactivate both alleles of the genes encoding critical signal-transduction proteins in the interferon signaling pathway. Researchers looked for mutant cells that expressed the interferon receptor (as evidenced by the cell's ability to bind radioactive interferon) but did not express GPRT in response to interferon and thus survived killing by 6-thioguanine when cells were cultured in the presence of interferon (Figure 14-10b). After many such interferon-nonresponding mutant cell lines were obtained, they were used to screen a genomic or cDNA library for the wild-type genes that complemented the mutated genes in nonrespond-ing cells, a technique called functional complementation (see Figure 9-20). In this case, mutant cells expressing the corresponding recombinant wild-type gene grew on HAT medium and were sensitive to killing by 6-thioguanine in the presence of interferon. That is, they acted like wild-type cells.

Cloning of the genes identified by this procedure led to recognition of two key signal-transduction proteins: a JAK tyrosine kinase and a STAT transcription factor. Subsequent work showed that one (sometimes two) of the four human

▲ EXPERIMENTAL FIGURE 14-11 Studies with mutant mice reveal that both the erythropoietin receptor (EpoR) and JAK2 are essential for development of erythrocytes. Mice in which both alleles of the EpoR or JAK2 gene are "knocked out" develop normally until embryonic day 13, at which time they begin to die of anemia due to the lack of erythrocyte-mediated transport of oxygen to the fetal organs. The red organ in the wild-type embryos (+/+) is the fetal liver, the major site of

JAK proteins and at least one of several STAT proteins are involved in signaling downstream from all cytokine receptors. To understand how JAK and STAT proteins function, we examine one of the best-understood cytokine signaling pathways, that downstream of the erythropoietin receptor.

Receptor-Associated JAK Kinases Activate STAT Transcription Factors Bound to a Cytokine Receptor

The JAK2 kinase is tightly bound to the cytosolic domain of the erythropoietin receptor (EpoR). Like the three other members of the JAK family of kinases, JAK2 contains an N-terminal receptor-binding domain, a C-terminal kinase domain that is normally poorly active catalytically, and a middle domain of unknown function. JAK2, erythropoietin, and the EpoR are all required for formation of adult-type erythrocytes, which normally begins at day 12 of embryonic development in mice. As Figure 14-11 shows, embryonic mice lacking functional genes encoding either the EpoR or JAK2 cannot form adult-type erythrocytes and eventually die owing to the inability to transport oxygen to the fetal organs.

As already noted, erythropoietin binds simultaneously to the extracellular domains of two EpoR monomers on the cell surface (see Figure 14-8). As a result, the associated JAKs are brought close enough together that one can phosphorylate the other on a critical tyrosine in the activation lip. As with other kinases, phosphorylation of the activation lip leads to a conformational change that reduces the Km for ATP or the substrate to be phosphorylated, thus increasing the kinase activity. One piece of evidence for this activation mechanism comes from study of a mutant JAK2 in which the critical tyrosine is mutated to phenylalanine. The mutant JAK2 binds normally to the EpoR but cannot be phosphorylated.

erythrocyte production at this developmental stage. The absence of color in the mutant embryos (—/—) indicates the absence of erythrocytes containing hemoglobin. Otherwise the mutant embryos appear normal, indicating that the main function of the EpoR and JAK2 in early mouse development is to support production of erythrocytes. [EpoR images from H. Wu et al., 1995, Cell 83:59; JAK2 images from H. Neubauer et al., 1998, Cell 93:307.]

Expression of this mutant JAK2 in erythroid cells in greater than normal amounts totally blocks EpoR signaling, as the mutant JAK2 blocks the function of the wild-type protein. This type of mutation, referred to as a dominant negative, causes loss of function even in cells that carry copies of the wild-type gene (Chapter 9).

Once the JAK kinases become activated, they phospho-rylate several tyrosine residues on the cytosolic domain of the receptor. certain of these phosphotyrosine residues then serve as binding sites for a group of transcription factors collectively termed STATs. All STAT proteins contain an N-terminal SH2 domain that binds to a phosphotyrosine in the receptor's cytosolic domain, a central DNA-binding domain, and a c-terminal domain with a critical tyrosine residue. Once a STAT is bound to the receptor, the C-terminal tyro-

▲ FIGURE 14-12 JAK-STAT signaling pathway. Following ligand binding to a cytokine receptor and activation of an associated JAK kinase, JAK phosphorylates several tyrosine residues on the receptor's cytosolic domain (see Figure 14-5, bottom). After an inactive monomeric STAT transcription factor binds to a phosphotyrosine in the receptor, it is phosphorylated by active JAK. Phosphorylated STATs spontaneously dissociate from the receptor and spontaneously dimerize. Because the STAT homodimer has two phosphotyrosine-SH2 domain interactions, whereas the receptor-STAT complex is stabilized by only one such interaction, phosphorylated STATs tend not to rebind to the receptor. The STAT dimer, which has two exposed nuclear-localization signals (NLS), moves into the nucleus, where it can bind to promoter sequences and activate transcription of target genes.

sine is phosphorylated by an associated JAK kinase (Figure 14-12). This arrangement ensures that in a particular cell only those STAT proteins with an SH2 domain that can bind to a particular receptor protein will be activated. A phosphorylated STAT dissociates spontaneously from the receptor, and two phosphorylated STAT proteins form a dimer in which the SH2 domain on each binds to the phos-photyrosine in the other. Because dimerization exposes the nuclear-localization signal (NLS), STAT dimers move into the nucleus, where they bind to specific enhancer sequences controlling target genes.

Different STATs activate different genes in different cells. In erythroid progenitors, for instance, stimulation by erythropoietin leads to activation of STAT5. The major protein induced by active STAT5 is Bcl-xL, which prevents the programmed cell death, or apoptosis, of these progenitors, allowing them to proliferate and differentiate into erythroid cells (see Figure 14-7). Indeed, mice lacking STAT5 are highly anemic because many of the erythroid progenitors undergo apoptosis even in the presence of high erythropoietin levels. Such mutant mice produce some erythrocytes and thus survive, because the erythropoietin receptor is linked to other anti-apoptotic pathways that do not involve STAT proteins (see Figure 14-9).

SH2 and PTB Domains Bind to Specific Sequences Surrounding Phosphotyrosine Residues

As noted earlier, many intracellular signal-transduction proteins contain an SH2 or PTB domain by which they bind to an activated receptor or other component of a signaling pathway containing a phosphotyrosine residue (see Figure 14-6). The SH2 domain derived its full name, the Src homology 2 domain, from its homology with a region in the prototypical cytosolic tyrosine kinase encoded by the src gene. The three-dimensional structures of SH2 domains in different proteins are very similar, but each binds to a distinct sequence of amino acids surrounding a phosphotyrosine residue. The unique amino acid sequence of each SH2 domain determines the specific phosphotyrosine residues it binds.

The SH2 domain of the Src tyrosine kinase, for example, binds strongly to any peptide containing a critical four-residue core sequence: phosphotyrosine-glutamic acid-glutamic acid-isoleucine (Figure 14-13). These four amino acids make intimate contact with the peptide-binding site in the Src SH2 domain. Binding resembles the insertion of a two-pronged "plug"—the phosphotyrosine and isoleucine side chains of the peptide—into a two-pronged "socket" in the SH2 domain. The two glutamic acids fit snugly onto the surface of the SH2 domain between the phosphotyrosine socket and the hydrophobic socket that accepts the isoleucine residue.

Variations in the hydrophobic socket in the SH2 domains of different STATs and other signal-transduction proteins allow them to bind to phosphotyrosines adjacent to different sequences, accounting for differences in their binding partners.

▲ FIGURE 14-13 Surface model of the SH2 domain from Src kinase bound to a phosphotyrosine-containing peptide. The peptide bound by this SH2 domain (gray) is shown in spacefill. The phosphotyrosine (Tyr0 and OPO3~, orange) and isoleucine (Ile3, orange) residues fit into a two-pronged socket on the surface of the SH2 domain; the two glutamate residues (Glu1, dark blue; Glu2, light blue) are bound to sites on the surface of the SH2 domain between the two sockets. Nonbinding residues on the target peptide are colored green. [See G. Waksman et al., 1993, Cell 72:779.]

The binding specificity of SH2 domains is largely determined by residues C-terminal to the phosphotyrosine in a target pep-tide. In contrast, the binding specificity of PTB domains is determined by specific residues five to eight residues N-terminal to a phosphotyrosine residue. Sometimes a PTB domain binds to a target peptide even if the tyrosine is not phosphorylated.

Signaling from Cytokine Receptors Is Modulated by Negative Signals

Signal-induced transcription of target genes for too long a period can be as dangerous for the cell as too little induction. Thus cells must be able to turn off a signaling pathway quickly unless the extracellular signal remains continuously present. In various progenitor cells, two classes of proteins serve to dampen signaling from cytokine receptors, one over the short term (minutes) and the other over longer periods of time.

Short-Term Regulation by SHP1 Phosphatase Mutant mice lacking ,SHP1 phosphatase die because of excess production of erythrocytes and several other types of blood cells. Analysis of these mutant mice offered the first suggestion that SHP1, a phosphotyrosine phosphatase, negatively regulates signaling from several types of cytokine receptors in several types of progenitor cells.

How SHP1 dampens cytokine signaling is depicted in Figure 14-14a. In addition to a phosphatase catalytic domain, SHP1 has two SH2 domains. When cells are not stimulated

(b) Signal blocking and protein degradation induced by SOCS proteins

Recruitment v>*. of E3 ubiquitin ligase

▲ FIGURE 14-14 Two mechanisms for terminating signal transduction from the erythropoietin receptor (EpoR).

(a) SHP1, a protein tyrosine phosphatase, is present in an inactive form in unstimulated cells. Binding of an SH2 domain in SHP1 to a particular phosphotyrosine in the activated receptor unmasks its phosphatase catalytic site and positions it near the phosphorylated tyrosine in the lip region of JAK2. Removal of the phosphate from this tyrosine inactivates the JAK kinase.

(b) SOCS proteins, whose expression is induced in erythropoietin-stimulated erythroid cells, inhibit or permanently terminate signaling over longer time periods. Binding of SOCS to phosphotyrosine residues on the EpoR or JAK2 blocks binding of other signaling proteins (left). The SOCS box can also target proteins such as JAK2 for degradation by the ubiquitin-proteasome pathway (right). Similar mechanisms regulate signaling from other cytokine receptors. [Part (a) adapted from

S. Constantinescu et al., 1999, Trends Endocrin. Metabol. 10:18;

part (b) adapted from B. T Kile and W. S. Alexander, 2001, Cell. Mol. Life

(b) Signal blocking and protein degradation induced by SOCS proteins

by a cytokine (are in the resting state), one of the SH2 domains physically binds to and inactivates the catalytic site in SHP1. In the stimulated state, however, this blocking SH2 domain binds to a specific phosphotyrosine residue in the activated receptor. The conformational change that accompanies this binding unmasks the SHP1 catalytic site and also brings it adjacent to the phosphotyrosine residue in the activation lip of the JAK associated with the receptor. By removing this phosphate, SHP1 inactivates the JAK, so that it can no longer phosphorylate the receptor or other substrates (e.g., STATs) unless additional cytokine molecules bind to cell-surface receptors, initiating a new round of signaling.

Long-Term Regulation by SOCS Proteins Among the genes whose transcription is induced by STAT proteins are those encoding a class of small proteins, termed sSOCS proteins, that terminate signaling from cytokine receptors. These negative regulators, also known as CIS proteins, act in two ways (Figure 14-14b). First, the SH2 domain in several SOCS proteins binds to phosphotyrosines on an activated receptor, preventing binding of other SH2-containing signaling proteins (e.g., STATs) and thus inhibiting receptor signaling. One SOCS protein, SOCS-1, also binds to the critical phos-photyrosine in the activation lip of activated JAK2 kinase, thereby inhibiting its catalytic activity. Second, all SOCS proteins contain a domain, called the ,SOCS box, that recruits components of E3 ubiquitin ligases (see Figure 3-13). As a result of binding SOCS-1, for instance, JAK2 becomes polyu-biquitinated and then degraded in proteasomes, thus permanently turning off all JAK2-mediated signaling pathways. The observation that proteasome inhibitors prolong JAK2 signal transduction supports this mechanism.

Studies with cultured mammalian cells have shown that the receptor for growth hormone, which belongs to the cy-tokine receptor superfamily, is down-regulated by another SOCS protein, SOCS-2. Strikingly, mice deficient in this SOCS protein grow significantly larger than their wild-type counterparts and have long bone lengths and proportionate enlargement of most organs. Thus SOCS proteins play an essential negative role in regulating intracellular signaling from the receptors for erythropoietin, growth hormone, and other cytokines.

Mutant Erythropoietin Receptor That Cannot Be Down-Regulated Leads to Increased Hematocrit

In normal adult men and women, the percentage of erythro-cytes in the blood (the hematocrit) is maintained very close to 45-47 percent. A drop in the hematocrit results in increased production of erythropoietin by the kidney. The elevated ery-thropoietin level causes more erythroid progenitors to undergo terminal proliferation and differentiation into mature erythrocytes, soon restoring the hematocrit to its normal level. In endurance sports, such as cross-country skiing, where oxygen transport to the muscles may become limiting, an excess of red blood cells may confer a competitive advantage. For this reason, use of supplemental erythropoietin to increase the hematocrit above the normal level is banned in many athletic competitions, and athletes are regularly tested for the presence of commercial recombinant erythropoietin in their blood and urine.

H Supplemental erythropoietin not only confers a possible competitive advantage but also can be dangerous. Too many red cells can cause the blood to become sluggish and clot in small blood vessels, especially in the brain. Several athletes who doped themselves with erythropoietin have died of a stroke while exercising.

Discovery of a mutant, unregulated erythropoietin receptor (EpoR) explained a suspicious situation in which a winner of three gold medals in Olympic cross-country skiing was found to have a hematocrit above 60 percent. Testing for ery-thropoietin in his blood and urine, however, revealed lower-than-normal amounts. Subsequent DNA analysis showed that the athlete was heterozygous for a mutation in the gene encoding the erythropoietin receptor. The mutant allele encoded a truncated receptor missing several of the tyrosines that normally become phosphorylated after stimulation by erythropoietin. As a consequence, the mutant receptor was able to activate STAT5 and other signaling proteins normally, but was unable to bind the negatively acting SHP1 phosphatase, which usually terminates signaling (see Figure 14-14a). Thus the very low level of erythropoietin produced by this athlete induced prolonged intracellular signaling in his erythroid progenitor cells, resulting in production of higher-than-normal numbers of erythrocytes. This example vividly illustrates the fine level of control over signaling from the erythropoietin receptor in the human body. I

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