Signal Transduction
Other proteins that regulate MAP kinase pathways
RKIP is an inhibitor of the Ras–ERK pathway.162 It binds to C-Raf (and weakly to MEK1, but not to B-Raf) and it prevents MEK activation. When phosphorylated by PKC, it is displaced from C-Raf, relieving this inhibition and so reinstating the Ras–ERK pathway and contributing to gene transcription through the AP-1 complex (see page 580).163
The 7TM receptor-linked -arrestins (see page 98) also act as scaffolds for the MAP kinase pathway. Unlike KSR1, the -arrestins appear to retain active signalling cassettes (comprising C-Raf, MEK, and ERKs 1 and 2 or JNK3) in the cytosol and thereby prevent phosphorylation of nuclear substrates.164
MP-1 localizes the ERK1 signalling cassette to late endosomes through an interaction with the endosomal p14 adaptor protein.165,166 Signalling from the receptor continues in the endosomal compartment (see below).167,168
Why are the signalling pathways so complicated?
And why are there so many apparently redundant components? In fact, not all pathways are long and complicated. Some, like the pathway involving Notch (Chapter 22) or the STAT proteins (see below) are relatively straightforward, entailing only the modification of a signalling protein at the membrane followed by its direct translocation into the nucleus. More generally, however, mechanisms tend to be complex and while there is no simple explanation, the reasons for complexity must stem from the need for cells to be able to sense multiple inputs, to commit to sets of appropriate responses and to conduct them in controlled and precise ways. In general:
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Growth factor signals must initiate and synchronize both transcriptional and metabolic events. This requires multiple effectors, both nuclear and cytosolic.
Signals need to propagate among and between different cellular compartments. (For example, receptors may be removed from the cell surface by endocytosis, processed in vesicular compartments and recycled back to the plasma membrane.)
A single signalling protein can only make contact with a limited number of effectors.
Signals must be amplified in order to exceed downstream threshold conditions and ensure effective responses.
Signals must be robust and their propagation should not rely on a single critical component.
A timely and measured response requires feedback circuits in order to maintain balanced and dynamic relationships between inputs and outputs.
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•Signals from different inputs must converge at sites of integration that determine appropriate output signals.
Beyond all this, one must recall that signal transduction pathways were never designed, but are the result of numerous rather messy trial and error processes. In the end, these proved, and continue to prove, to be of benefit to the organisms of which they form a part.
Termination of the ERK response
The ability to attenuate and to terminate signals initiated by growth factors is crucial, and there are numerous ways by which this is achieved. We limit the discussion to events affecting the receptors and the adaptors and effectors that bind to them.
First, there are the phosphatases, including PTP1B, SHP1/2, and DEP1, that strip phosphotyrosine residues. These, as well as others that inactivate ERK, are further discussed in Chapter 21. Secondly, phosphorylation of the guanine nucleotide exchange factor Sos1 by ERKs 1 and 2 reduces its affinity for Grb2, so suppressing the Ras signalling pathway.169,170 Downstream of ERK, RSK can also phosphorylate Sos1 (but not Sos2, which lacks several serine/threonine phosphorylation sites).119
A third mechanism of negative feedback is the removal of cell surface receptors by endocytosis. In the case of EGF, endocytosis following low-level stimulation actually induces a burst of signalling before suppressing it, due to a contribution from the internalized receptor.168 Signalling only ceases when the receptors are degraded by acid hydrolases in late endosomes. Phosphorylated receptors are preferentially endocytosed and directed by vesicular transport to the late endosomal compartment through the actions of Grb2 and Cbl
(an E3-ubiquitin ligase) (see Figure 12.21). Both bind to receptors through their (atypical) SH2 domains and both contribute to the clustering of receptors and their uptake into clathrin-coated vesicles.171,172 Cbl may also act as a handle, to prevent recycling of the activated EGFR to the plasma membrane.
A family of MAP kinase-related proteins
Once ERKs 1 and 2 were cloned, it became apparent that they are members of a substantial family, the MAP kinases. Based on sequence analysis and the composition of their activation segments, they may be classified into five groups, each operating in different signal transduction pathways (see Figure 12.22 and Table 12.2).
•ERK1 and ERK2 are the ‘prototypic’ or ‘classical’ MAP kinases, operating mainly in mitogen activated signal transduction pathways.
•ERK3 and ERK4 are distinguished from other ERKs by the absence of a tyrosine in the activation segment. Thus they possess only a single (serine)
Cbl and monoubiquitylation. Ubiquitylation is controlled by ubiquitin activating (E1), conjugating (E2), and ligase (E3) enzymes. It results in the attachment of the small ubiquitin protein (76 residues) to a substrate (for more detail see page 467). Ubiquitin, and ubiquitinlike peptides, are now recognized as general signalling devices with have several roles.173 Attachment of four
or more ubiquitins, polyubiquitylation, is a recognition signal for destruction by the proteasome; monoubiquitylation is
a signal for endocytosis and a signal in histone regulation. In the endosome, ubiquitin serves as a molecular signature on trafficking cargoes. Cbl, an E3-ligase, catalyses ubiquitylation of the EGFR, so that it is recognized by proteins bearing ubiquitin-binding domains. For instance, the adaptor protein Eps15 has two ubiquitininteracting motifs that interact with protein sorting machinery.
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Fig 12.21 Removal of the EGFR from the cell surface and sorting into the lysosome pathway.
Activated EGF receptors are recognized by Cbl which binds either directly through a phosphotyrosine-binding motif or by interaction with the SH3 domain of Grb2. Ubiquitylation by Cbl acts as a sorting signal, directing the receptor into the lysosomal pathway for degradation. The receptor–Cbl complex is recognized by CIN85 and endophilin, which couple the receptor to a group of proteins that includes the endocytic adaptor AP-2. Clathrin monomers are then recruited and the active EGFRs accumulate in clathrin-coated membrane pits which pinch off from the plasma membrane as endocytic vesicles. Within the intracellular network of vesicular transport pathways, the receptors progress through the early and late endosomes to the lysosome and are destroyed.
Cycloheximide, a product of the bacterium
Streptomyces griseus, is an inhibitor of protein biosynthesis in eukaryotic organisms. It interferes with the activity of peptidyl transferase activity, thus blocking translational elongation.
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phosphorylation site in the motif SEG. Little is known about their upstream regulators and substrates. ERK3 has two gene variants, and .94
ERK5 (originally called Big MAP kinase or BMK1) is activated by mitogens, but its C-terminal domain gives rise to a protein twice the size of ERKs
1 and 2. It has transcriptional activity, either on its own, binding direct to DNA, or through its association with other transcription factors (for instance the AP-1 complex). It operates in a similar pathway as ERKs 1
and 2, and has many substrates in common. It has a role in cardiovascular development and neural differentiation.181
JNK and SAPK phosphorylate c-Jun174,175 and are activated in response to ‘stress’.176 They emerged in experiments in which rat livers were challenged by injection of cycloheximide.177 Activating factors include growth factor
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Fig 12.22 Parallel MAP kinase pathways.
The MAP kinases are classified in three groups based on the identity of the intermediate residue in their dual phosphorylation motifs (TEY, TGY, or TPY). This classification also defines the ERK, JNK, and p38 signal transduction pathways, each having unique upstream activators. The ERK pathway acts in response to mitogens, whereas the p38 and JNK pathways respond to stress and inflammatory cytokines.
deprivation, UV irradiation, or treatment with inflammatory cytokines (IL- 1 , TNF). The JNKs include JNK1, 2, and 3.178
•p38 kinase came to light in genetic deletion studies in yeast (S. cerevisiae) as a kinase involved in the generation of glycerol in response to osmotic stress.179 HOG1 induces expression of glycerol-3-phosphate dehydrogenase, related to glycerol synthesis.180 In mammalian cells, it is also activated in response to stress stimuli. p38/HOG is now generally referred to as p38 kinase, of which there are four variants: p38 , , , and .94
Each of the pathways shown in Figure 12.22 involves a kinase cascade, comprising a MAP3K and MAP2K and culminating in the phosphorylation and activation of the particular MAP kinase. Each contains a dual phosphorylation site, TEY, TPY, or TGY, (with the exception of ERK3 and ERK4, see above). These parallel pathways of activation may operate individually or in combination to initiate specific patterns of gene expression. Although it is generally accepted that the ERK pathway responds to mitogen stimulation, and the JNK and
p38 pathways to stress and inflammation, cross-talk undoubtedly occurs. We elaborate further on the roles of JNK and p38 and their role in the regulation of the innate immune response (inflammation) in Chapter 16 (see page 493).
There is still an awful lot to learn about Ras. As our knowledge of Ras and the pathways that it regulates have advanced, so its involvements have
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become more complex and entangled. Ras is not merely an activator of cell growth. Indeed, in some cells it causes growth inhibition and differentiation. In others it blocks differentiation. It has become apparent that it is a regulator of multiple cell functions that depend on the cell, the state of the cell, the activation state of other GTPases and possibly the identity of the Ras isotype (N-, K-, or H-). What’s more, its relationship to cell transformation is not simply a matter of cause and effect. As examples:
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Oncogenic Ras only induces transformation if the recipient cell has already endured a number of mutations in tumour suppressor genes such as p53 or Rb. Otherwise its introduction results in apoptosis (see page 306). Most of the experimental work on Ras has been carried out on rodent fibroblast cell lines that are far more susceptible to transformation than the epithelial cells in which most (human) Ras-related tumours occur. More than this, the phenotypes of the transformed cells are very different. Ras-transformed (human breast) epithelial cells are characterized by disruption of the adherens junctions and the appearance of stress fibres and focal adhesions (see Figure 13.15, page 399) but transformed fibroblasts are associated with a loss of stress fibres. While constitutively activated mutants of Ras, or its effector Raf, can induce the transformed phenotype in mouse fibroblasts, only Ras can induce transformation of rat intestinal epithelial cells.
Due to the availability of antibodies, mutants, etc., most investigations have concentrated on H-Ras. However, although the human Ras genes N, H, and K are very similar, and in many experimental situations appear to function in the same way, there is no reason to believe that their actions are identical. The conservation of three ras genes in vertebrate evolution begs the question of whether or not the gene products have specific functions. Although the data are so far very sketchy, the embryonic lethality of K-ras (but not of N- or H-ras) supports the idea of nonredundancy of function.
In this chapter we have indicated the role of Ras as an activator of ERK, but this is not the only pathway implicated in its regulation of cell proliferation. In Chapter 18 we consider Ras as one of a number of activators of PI 3-kinase
and the consequent activation of protein kinase B. There are several additional (and also potential) effector pathways through which the effects of activated Ras can operate.
For a more comprehensive discussion of these questions, see Shields et al.182
MAP kinases in other organisms
Pathways regulated by MAP kinases are present in all eukaryotic organisms.183 In yeast (S. cerevisiae), processes regulated by MAP kinases include mating, sporulation, maintenance of cell wall integrity, invasive growth, pseudohyphal
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