Signalling Pathways Operated by Receptor Protein Tyrosine Kinases
PLC , the only isoform directly activated by the EGF receptor, possesses an SH2 domain (see page 299).38 Further evidence was provided by the finding that SH2 domains bind to phosphotyrosine ligands, including those present in activated EGF receptors.62 The basic structure and properties of SH2 domains are described in Chapter 24 (see page 768).
Another domain, structurally unrelated, that binds to phosphotyrosines, also present in many signalling molecules, is the PTB (phosphotyrosine-binding) domain (see page 771). This was first identified in the adaptor protein Shc, which also contains an SH2 domain.63 These protein interaction domains recognize phosphotyrosines within specific oligopeptide motifs. The optimal sequences of these short stretches are similar for the same type of domain, but those recognized by SH2 and PTB domains differ considerably.64,65
Many proteins having SH2 or PTB domains (or both) associate with rPTKs to initiate the assembly of signalling complexes (Figure 12.6 and Figure 12.7). Some of these proteins themselves then become phosphorylated as a result of this association. For PLC , this is a necessary activation step, but it is not clear whether this is the case for other effectors.
The human genome has 109 proteins predicted to have SH2 domains and 44 with PTB domains.24 Some of these occur as tandem pairs (Figure 12.6). Most of the proteins that have these domains are either adaptors with no catalytic activity, or effectors such as phospholipases and protein/lipid kinases; others are transcription factors.
The proteins that associate with EGF receptors exhibit preferences for the sequence of amino acid residues in the close vicinity of the phosphorylated tyrosine. In consequence, for a given motif some proteins bind more tightly than others. This enables receptors to signal through panels of SH2-containing proteins. For example, a screen of SH2 and PTB domains (66 phosphopeptides) derived from the ErbB family, revealed that some could bind a wide range
of effectors (such as PI 3-kinase, phospholipase C , the tyrosine kinase Syk, the adaptors Shc1 and Crk, and RasGAP), while others are more specific.24 Significantly, for the EGFR and ErbB2 (but not ErbB3), the interaction becomes less selective as the concentration (in the assay) of the phosphopeptide ligand increases. Thus, experimental over-expression of these rPTKs broadens the
possible range of downstream signalling pathways. High expression of the EGFR or ErbB2 (but not ErbB3) certainly occurs in breast cancer (see Chapter 23).
Branching of the signalling pathway
A number of signal transduction pathways branch out from the signalling complex (Figure 12.8). Two such branches are described in the following paragraphs and others are considered later (STATs in Chapter 17, PI 3-kinase in Chapter 18).
Gag: glycosylated antigen, the gene encoding the internal capsid of the viral particle.
Crk: C10 regulator of kinase. This is an SH2 and SH3 domain-containing adaptor protein
and is implicated in pathogenesis of chronic myelogenous leukemia.
p47gag-crk is a good
example of how viruses disrupt genes resulting in chimeric proteins.
In this case, viral Gag sequences are fused to the cellular CrkL. The mammalian cells express the gene product once it is inserted into their genome.
The affinity (KD) of SH2 or PTB domains for tyrosinephosphorylated peptides is not particularly strong, of the order of 2 mol L 1. In comparison, the affinity of growth factors for their receptors is typically KD 0.1 mol L 1. As a consequence,
the interactions have a half-life of less than 10 s, so that within the duration of receptor responses (minutes), numerous adaptor and
effector complexes form, detach and reform.24 This dynamic interplay allows tyrosine phosphatases to intervene, removing the phosphoryl groups from the rPTKs (see Chapter 21).
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Fig 12.6 Domain architecture of proteins that associate with phosphorylated tyrosine kinase-containing receptors. Most of the effectors and adaptors and all docking proteins are also substrates of the receptor kinases. Phosphorylated docking proteins offer novel phosphotyrosine docking sites for yet other adaptors and effectors. Many of the proteins presented in this figure are discussed elsewhere in this book. Abbreviations are listed at the end of the chapter.
The PLC –PKC signal transduction pathway
Among the activities set in train by activation of the EGF and PDGF receptors is the generation of DAG and IP3 by PLC . DAG remains in the membrane and acts as a stimulus for PKC. The consequence is the transformation of a phosphotyrosine signal, through activation of PLC , into a phosphoserine/ phosphothreonine signal.
One of the first substrates of PKC is the EGF receptor itself. It becomes phosphorylated on a serine situated close to the transmembrane domain,
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Fig 12.7 Formation of receptor signalling complexes. SH2 and PTB domains bind to specific motifs containing a phosphotyrosine. (1) The SH3 domain of Grb2 attaches to a proline rich sequence in Sos, a guanine nucleotide exchange factor. (2) The SH2 domain of Grb2 binds the tyrosine phosphorylated EGFR, leading to recruitment of the adaptor/effector complex to the membrane, there to find its target, Ras. (3) An alternative route
for recruitment of Grb2 occurs through the intervention of Shc. The PTB domain of Shc binds the tyrosine phosphorylated EGF. (4) This leads to its phosphorylation, (5) creating a novel binding site for Grb2. The discovery of Grb2 as an adaptor and Sos as a guanine nucleotide exchange factor for Ras is described below on page 332. Key residues within the binding motifs are shown in red in the table.
causing its inactivation. Although numerous proteins have proved to be substrates of the PKC enzymes, we still lack full understanding of how these kinases determine the changes in gene transcription that occur after stimulation. As discussed in Chapter 19 (see page 578), one consequence of PKC activation is actually dephosphorylation of c-Jun, a component of the inducible transcription factor AP-1.
The Ras signalling pathway
From the tyrosine kinase to Ras
For 40 years it has been known that infection of rats with murine leukaemia viruses can provoke the formation of a sarcoma.66 A major advance was
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Fig 12.8 Branching of the signal transduction pathways. Following activation of a receptor PTK, several signal transduction pathways can be activated. Five are indicated.
the discovery that the Harvey murine sarcoma virus encodes a persistently activated form of H-ras in which valine is substituted for glycine at position 12 (see page 106). Expression of this mutant in quiescent rodent fibroblasts results in altered cell morphology, stimulation of DNA synthesis and cell proliferation.67 When over-expressed, (normal) H-Ras also induces oncogenic transformation,68 as does micro-injection of the mutant protein.69 Conversely, injection of neutralizing antibodies to inhibit normal Ras function reverses cell transformation.70 Furthermore, stimulation of quiescent cells with serum or with growth factors promotes the binding of GTP to Ras.71 These findings showed that Ras is an important component in the signalling pathways that regulate cell proliferation, but how it fits into the known pathways emanating from growth factor receptors remained unclear for a considerable time. The first clues came from genetic analysis of signal transduction pathways that operate in the invertebrates Drosophila melanogaster and Caenorhabditis elegans (in plain English, flies and worms).
Photoreceptor development in the fruit fly
The compound eyes of insects are arrays of small hexagonal units called ommatidia. The fruit fly has approximately 800 of these ‘small eyes’, arranged as shown in Figure 12.9. Each is composed of eight photoreceptor cells (R1–R8) and 12 accessory cells. On the basis of their morphology, order of development, axon projection pattern, and spectral sensitivity, the photoreceptor cells can be classified into three functional classes. R8 is the first to appear, followed
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Fig 12.9 The sevenless mutation in fly eyes. The events leading to the development of cell R7 in Drosophila eye have provided a key to understanding the pathways initiated by rPTKs. Genes acting downstream of the sevenless receptor were revealed by screening for mutations that affect the development of cell R7. (a) A scanning electron microscope image showing the geometrical arrangement of ommatidia, units of eight photoreceptor cells. (b), (c) Thin sections, representative of cuts through section B–B in the drawing. Note that in (b), taken from a wild-type fly, seven cells are evident, while in (c), taken from a fly having the sevenless mutation, there are only six. The drawing illustrates the basic anatomy of a single ommatidial unit in longitudinal section. Sections cut at A, B, and C are shown in transverse section on the right. Since two of the cells, R7 and R8, do not extend the full length of the ommatidial unit, the transverse sections B–B and C–C only reveal seven, not all eight cells. For further information, see http://flybase.bio.indiana. edu/ Picture adapted from Dickson and Hafen.73
by R1–R6 and then R7. The photosensitive pigment resides in a stack of membranes, the rhabdomere. The larger rhabdomeres of cells R1–R6 are arranged as a trapezoid surrounding the rhabdomeres of cells R7 and R8. The R8 rhabdomere is located below R7 (Figure 12.9). The development of R7 requires the products of two genes, sevenless (sev) and bride-of-sevenless (boss). The phenotypes generated by loss-of-function mutations in either of these genes are identical, R7 failing to initiate neuronal development. These mutations are readily detected in a behavioural test. Given a choice between a green and a UV light, normal (WT) flies will move rapidly towards
the UV source.72 Failure to develop cell R7, the last of the photoreceptor cells to be added to the ommatidial cluster, correlates with the lack of this fast phototactic response and the flies move towards the green light.72
While the sev product is required only in the R7 precursor, boss function must be expressed in the developing R8. Cloning revealed the Boss product as a
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100 kDa glycoprotein with seven transmembrane spans that is related to the metabotropic receptors (see page 63).74 Although ultimately expressed on all of the photoreceptor cells, at the time that R7 is being specified it is only present on the oldest, R8.75 The product of the sev gene is an rPTK (member of the insulin receptor family).76 Evidence for direct interaction between these
two gene products came with the demonstration that cultured cells expressing the boss product tend to form aggregates with cells expressing Sev.75
It is now understood that the binding of Boss (the ligand) to Sev (the rPTK) leads to the activation of kinase activity and that this ultimately determines the fate of R7 as a neuronal cell. Since a reduction in the gene dosage of the fly Ras1 impairs signalling by Sev, and persistent activation of Ras1 obviates the need for the boss and sev gene products, it follows that the activation of Ras1 is an early consequence of Sev activity.77
Further genetic screens of flies expressing constitutively activated Sev led to the identification Drk and Sos as intermediate components of this pathway (see column 1 of Figure 12.10). The Sos protein shows substantial homology with the yeast CDC25 gene product, a guanine nucleotide exchange factor for RAS.78 Whereas reduction in the gene dosages of Drk and Sos impairs the
Fig 12.10 Comparison of signalling pathways activated by a receptor protein tyrosine kinase in species from different phyla. The striking homologies that exist between the proteins operating downstream of rPTKs in distant phyla has enabled the elucidation of the EGF receptor pathway.
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signal from constitutively activated Sev, there is no effect on signalling from constitutively activated Ras. Thus, in the pathway of activation, this places the functions of the Drk and Sos products into positions intermediate between Sev and Ras. The Drk gene codes for a small protein having no catalytic activity, but consists exclusively of Src homology domains, two SH3 flanking a single SH2 domain. An adaptor, it binds to the tyrosine-phosphorylated receptor and links it to the proline-rich domains of Sos.79
Vulval cell development in worms
In the nematode C. elegans, a similar pathway of activation involving autophosphorylation of an rPTK leads to activation of the GTPase Let60, a homologue of Ras (column 2 of Figure 12.10). This determines the development of vulval cells (Figure 12.11). Again, these proteins were first identified by genetic analysis of mutants, namely lethal mutations (let), mutations affecting vulval development (sem, sex muscle mutants) and alterations in cell lineage (lin, lineage mutants).80 They constitute the
components of a signal transduction pathway based on Lin-3 (a product of the anchor cell), Let-23 (a tyrosine kinase receptor of the p6 p cell), and Sem-5 that associates with a (Sos-like) guanine nucleotide exchange protein. This brings about nucleotide exchange on Let-60 (Figure 12.10). Lin-3 and Let-23 are, respectively, members of the neuregulin (ligand) and ErbB (receptor) family.
More than 20 000 individual species of roundworm in the phylum Nematoda
(named from the Greek for ‘thread-like’) have so far been described, though it has been suggested that there might be more than
500 000. They inhabit all terrains: sea and fresh water, the polar regions, the tropics, and deep oceanic trenches. A large proportion are parasitic, including pathogenic forms that affect plants and animals.
Fig 12.11 Vulval development in C. elegans. Because it is a relative simple structure, formed from just a few cells, the vulva is well suited for the genetic analysis of cell differentiation during embryological development. It is the product of just three cell lineages, the descendents of cells p5.p, p6.p, and p7.p. Development is initiated by a signal from the anchor cell that lies adjacent to p6.p. The ligand, lin-3 (homologue of EGF), produced by the anchor cell, binds its receptor Let-23 (homologous to the EGF-R) on the surface of cell p6.p. Cell p6.p in turn releases signals to its neighbours, p5.p and p7.p,
and initiates a sequence of events involving the MAP kinase pathway, which determines the fate of these cells as components of vulval tissue. For more information, consult http://www.wormbook.org/
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Neuregulin 1 (NRG1) and its receptors ErbB are implicated in the pathophysiology of schizophrenia. Among NRG1 receptors, ErbB4 plays crucial roles in neural development and in the modulation of
NMDA receptor signalling. In the prefrontal cortex of schizophrenic individuals, there is a marked increase in NRG1induced activation of ErbB4, though expression appears unaltered. NRG1 stimulation suppresses NMDA receptor activation and this is more pronounced in schizophrenic subjects.81
hSos comes in two flavours, 1 and 2. They both interact through their proline-rich C-termini with the SH3 domains
of Grb2. The binding affinity of hSos2 for Grb2 is significantly higher than that of hSos1.87
Warning: we stress again that investigations using high-expression vectors can reveal signal transduction pathways that do not
necessarily operate under physiological conditions.
The hermaphroditic C. elegans is a free-living roundworm, 1 mm in length, that inhabits temperate soils. It is used extensively as a model organism. The entire genome has been sequenced, and the developmental fate of every one of the 959 somatic cells is known. When crowded or starved of food, C. elegans enter a larval stage called the dauer state (see page 608). Dauer larvae are resistant to stress and do not undergo ageing.
In both worms and flies, the Ras protein acts as a switch that determines cell fate. In C. elegans, the activation of Ras determines the formation of vulval as opposed to hypodermal (skin) cells. In Drosophila photoreceptors, the
activation of Ras determines the development of R7 as a neuronal as opposed to a cone cell. In both cases, Ras proteins operate downstream of rPTKs that are activated by cell–cell interactions.
Regulation of Ras in vertebrates
The elucidation of the Ras pathway in vertebrates was based on the identification of proteins having sequence homologies with those present in Drosophila and C. elegans (column 3 of Figure 12.10).82–84 Expression or microinjection of these proteins (and appropriate reagents such as peptides,
antibodies, etc.) were used to restore or modulate the activity of this pathway in cells derived from mammals, flies, or worms, bearing loss-of-function mutations. A vertebrate protein Grb2, lacking catalytic activities but having SH2 and SH3 domains, was found to be capable of restoring function in Sem-5 deficient mutants. In addition, Grb2 was found to associate with a protein that is recognized by an antibody raised against the Drosophila protein, Sos. In this way the sequence of events became apparent. Grb2 is an adaptor protein, linking the phosphorylated tyrosine kinase receptor to the guanine nucleotide exchanger in vertebrates (Figure 12.12). The mammalian Sos homologue, hSos, is likewise a guanine nucleotide exchange factor which interacts with Ras.85 Grb2 is composed exclusively of Src homology domains, one SH2 flanked
by two SH3 domains, similar to the Drosophila adaptor protein Drk. Because of the nature of the interaction of SH3 with proline-rich sequences (see Chapter 24), it is likely that Grb2 and Sos remain associated even under nonstimulating conditions. The main effect of receptor activation is to ensure the recruitment of the Grb2/Sos complex to the plasma membrane.86
The adaptor protein Shc binds through its PTB domain to phosphorylated receptors and, on phosphorylation, becomes an indirect docking site for Grb2/Sos (see Figure 12.7). A third component, Gab1, may also be involved upstream in the relay of receptor signalling. This large docking protein contains multiple tyrosines that are directly phosphorylated by activated EGF and insulin receptors. High-level expression of Gab1 enhances cell growth and facilitates cell transformation.88
It is possible that yet other adaptor/docking proteins will come to light, but the principle of action remains the same. Their roles may vary between cell
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Fig 12.12 Regulation of the Ras-MAP kinase pathway by receptor protein tyrosine kinases. The adaptor Grb2, in association with Sos, attaches to the tyrosine-phosphorylated receptor through its SH2 domains. This brings the Grb2/Sos complex into the vicinity of the membrane to catalyse guanine nucleotide exchange on Ras.
The activated Ras associates with the serine/threonine kinase C-Raf. Its presence at the membrane results in activation and then phosphorylation of the dual-specificity kinase MEK1, which in turn phosphorylates ERK2 on both tyrosine and threonine. Dimerization allows ERK2 to interact with proteins that guide it to the nucleus. Protein–protein interaction domains (RBD and CRD: see text) are in indicated in yellow letters.
types or may depend on the stimulus and its timing. It may also depend on the number of receptors, the concentration of the ligand and the cytosolic concentration of the different adaptors.
As already pointed out (page 108), the Ras-GTPase activating protein RasGAP also contains two SH2 domains and it too binds to phosphotyrosines on activated receptors. It is also a component of the signalling complex that assembles on activated PDGF receptors. It remains unclear what, if any, role this association of GAP with the receptor has in relaying signals. For instance,
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