Signal Transduction
MEK is the substrate of anthrax lethal factor, LF, one of several toxins produced by Bacillus anthracis. It resembles a metalloprotease. By cleaving the N-terminal region of MEKs 1 and 2, LF inhibits their kinase activity and prevents signal transmission through the ERK
pathway.92,93 Whether this is the cause of lethality in anthrax infection is not clear.
cells that express a mutant of the PDGF receptor that is unable to bind RasGAP, manifest normal activation of Ras.89
From Ras to MAP kinase and the activation of transcription
The events following the activation of mammalian Ras lead to the activation of a series of kinases culminating in ERK (extracellular signal regulated kinase). ERK was originally recovered as a serine/threonine phosphorylating activity present in the cytosol of EGF-treated cells and given the name mitogenactivated protein kinase, MAP kinase, or MAPK.90 (Of the five isoforms, ERKs
1, 2, and 5 contribute to the mitogen signal transduction pathway described here (Table 12.2 and see page 335). Once activated, ERK can enter the nucleus to activate early response genes. All this operates quite independent of second messengers and, in some cell types, cyclic AMP actually opposes it (see below).91 The earlier steps in the pathway involve the phosphorylation of each target kinase on its activation segment (Figure 12.12).
The immediate activator of ERK is MEK (MAP kinase-ERK kinase,
Figure 12.10). The MEKs comprise seven members, of which only MEKs 1, 2, and 5 are involved in the activation of ERK. MEKs are notable in that they act as ‘dual-specificity protein kinases’, phosphorylating ERKs 1 and 2 on both a threonine and a tyrosine residue. These are present in the target sequence LTEYVATRWYRAPE (Table 12.2), which constitutes the activation segment. To date, the ERKs appears to be the unique substrate for phosphorylation by MEKs 1 and 2, indicating a particularly high level of specificity.94
Upstream of MEK, the first kinase in the cascade is C-Raf (also described as MAP-kinase-kinase-kinase or MAP3K, Figure 12.10). Like Ras, C-Raf was initially identified as an oncogene product (v-raf).95 It phosphorylates MEK
at two serine residues in the activation segment, giving rise to a catalytically competent kinase. The subsequent finding that activated Ras recruits C-Raf to the membrane and in consequence brings about kinase activation, links ERK with the Ras pathway.96–98 Both the Ras-binding domain (RBD) and the cysteine-rich domain (CRD), present in the N-terminal region of C-Raf, are instrumental for this function. Full activation also requires association with activated Ras.99,100 A mutant form of C-Raf, endowed with a C-terminal -Caax box that acts as a site for prenylation (page 105), and which is permanently associated with the plasma membrane, can instigate the downstream events independently of Ras (at least in part).101
Activation of C-Raf is complicated and still not clearly understood. It is associated with several proteins, among which are the serine/threonine phosphatase PP2A, the heat shock protein Hsp90, the scaffold protein 14-3-3, and the cochaperone Hsp50/Cdc37.102 An essential feature is that the
334
Signalling Pathways Operated by Receptor Protein Tyrosine Kinases
Table 12.2 Mammalian MAP kinases (human unless otherwise indicated)
MAP kinase |
Other names |
P site motif in |
% Seq. ident. |
UniProtKB/SwissProt |
|
|
activation segment |
to ERK2 |
code |
|
|
|
|
|
ERK1 |
MK03, p44 MAPK |
TEYVATRWYRAPE |
88 |
P27361 |
|
|
|
|
|
ERK2 |
MK01, p42 MAPK |
TEYVATRWYRAPE |
100 |
P28482 |
|
|
|
|
|
ERK3 |
MK06-Rat |
SEGLVTKWYRSPR |
43 |
P27704 |
|
|
|
|
|
ERK3 |
MK06, p97 MAPK |
SEGLVTKWYRSPR |
42 |
Q16659 |
|
|
|
|
|
ERK4 |
MK04, p63 MAPK |
SEGLVTKWYRSPR |
42 |
P31152 |
|
|
|
|
|
ERK5 |
MK07, BMK1 |
TEYVATRWYRAPE |
– |
Q13164 |
|
|
|
|
|
ERK7 |
MK15-Rat |
TEYVATRWYRAPE |
– |
Q9Z2A6 |
|
|
|
|
|
JNK1 |
MK08, SAPK |
TEYVVTRYYRAPE |
40 |
P45983 |
|
|
|
|
|
JNK2 |
MK09, SAPK |
TEYVVTRYYRAPE |
41 |
P45984 |
|
|
|
|
|
JNK3 |
MK10, SAPK |
TEYVVTRYYRAPE |
40 |
P53779 |
|
|
|
|
|
P38 |
MK14, CSBP, p38 |
TGYVATRWYRAPE |
50 |
P16539 |
|
|
|
|
|
P38 |
MK11, p38-2 |
TGYVATRWYRAPE |
47 |
Q15759 |
|
|
|
|
|
P38 |
MK12, ERK6, SAPK3 |
TGYVVTRWYRAPE |
44 |
P53778 |
|
|
|
|
|
P38 |
MK13, SAPK4 |
TGYVVTRWYRAPE |
42 |
O15264 |
Adapted from Chen et al.94
N-terminal region, comprising the CRD and RBD domains, hinders the activity of the catalytic domain (CR3). Removal of this restraint requires a number
of modifications, of which two will be mentioned. C-Raf is maintained in its inactive state by a locked conformation imposed by 14-3-3, tightly bound at two phosphoserine residues. The association of C-Raf with RasGTP and the plasma membrane requires dephosphorylation of a serine residue by
PP2A103 and this enables it to escape the inhibitory constraint of 14-3-3. It also renders it susceptible to phosphorylation at other residues, especially in the negatively charged region adjacent to the kinase domain (see Figure 12.13) and in the activation segment. The kinases that mediate these phosphorylations have yet to be identified. B-Raf lacks the N-region phosphorylation sites and only requires phosphorylation at the activation segment for full catalytic activity. Essential to all this is that 14-3-3 remains attached.104
Raf genes
The raf oncogenes came to light as acquired oncogenes present in the murine retrovirus 3611-MSV105 and the avian retrovirus Mill-Hill 2.106 Homologues
335
Signal Transduction
Fig 12.13 Domain architecture of the Raf proteins. The Raf isoforms, A-, B- and C-Raf, share three conserved regions, CR1, CR2, and CR3 (kinase domain, red, with two phosphorylation sites in the activation segment). CR1 contains the Ras binding (RBD) and the cysteine-rich (CRD) domains, both necessary for membrane recruitment. CR2 contains one of the 14-3-3 phosphoserine binding sites, the other is in the C-terminal region. Note that B-Raf differs with respect to the acidic regulatory region between CR2 and CR3, lacking a tyrosine which is replaced by aspartate (D448) and the S445 being constitutively phosphorylated. The V559E mutation in the activation segment of B-Raf, which renders the kinase highly active, is coloured green. Adapted from Wellbrock et al.107
of these proteins are present in Drosophila (D-raf) and C. elegans (lin-45). The mammalian genome contains three raf genes, of which C-Raf (also known as raf-1) is the most abundantly expressed and is present in all tissues.
A- and B-Raf are also widely expressed but, apart from neural tissues, at lower levels. The Raf proteins share a common architecture (Figure 12.13) comprising three main regions. The CR1 region contains the Ras binding domain (RBD) and a cysteine-rich domain (CRD), both needed for membrane localization and activation. CR2 has a 14-3-3 protein binding site, and CR3 is the kinase domain.107 Despite the fact that the different Raf proteins show much sequence similarity and all activate the same MEKs, mouse knock-outs indicate that they are not functionally redundant.102
The raf oncogene
Mutations in B-raf (but not in A- or C-raf), have been detected in melanoma, thyroid, colorectal, and ovarian cancers (MIM 164757). There are more than 45 mutations, of which the most common render the kinase constitutively active (gain-of-function, see page 307). Of these a valine to glutamate substitution (V599E) in the activation segment is prevalent108 (Figure 12.13). As
a consequence, the activation segment folds away from the P-loop to facilitate access of substrate without further need for phosphorylation. In a MEK1
336