ACTIVATING RAS AND MAPK
One of the central pathways in eukaryotic cell signalling is that linking activated RTKs to RAS and the mitogen-activated protein kinase (MAPK) pathway (Fig. 1).
1.A major signalling pathway from plasma membrane to nucleus.
An essential component of this sequence is an adaptor protein, GRB2 (growth factor receptor-bound protein 2). Made up of an SH2 domain flanked by two SH3 domains, GRB2 is recruited to activated RTKs and its N-terminal SH3 domain binds to the guanine exchange factor SOS. Activation of RAS requires the dissociation of GDP, which is achieved by SOS that, in addition, draws RAS to the plasma membrane. These SOS-mediated effects change the conformation of RAS to promote interaction with the next member of the chain, RAF1. There is no evidence implicating SOS mutations in cancer but they do occur in some hereditary conditions, notably Noonan syndrome, which gives rise to a form of dwarfism.
Three subsequent protein associations cascade to the nucleus: RAF1 (activated by RAS) interacts with MAPKK, MAPKK with MAPK and, finally, MAPK phosphorylates transcription factors (TF in Fig. 1; e.g. ERK1). After phosphorylation by MAPK, transcription factors can enter the nucleus and bind directly to DNA. RAS is the prototype of a superfamily of molecular switches comprising more than 50 members in five sub-families (Table 1).
The MAPK cascade that is activated by RAS is also conserved across all organisms as a central mechanism for signal transduction, and there are several families of MAPK pathways that respond to different types of agonist (hormones, cytokines, stress signals, etc.) (Fig. 2).
2.Conserved MAPK pathways in mammalian cells
These enzymes are further examples of molecular switches: they are switched on by phosphorylation and they remain activated, independent of contact with their upstream activator until they are de-phosphorylated by the action of a phosphatase. The sequential interactions between MAP3Ks, MAP2Ks and MAPKs define linear routes leading to the activation of transcription factors. In addition, ‘cross-talk’ can occur between some components of distinct pathways. Mammalian cells have a variety of other signalling pathways in addition to the MAPK family and which of these are activated depends not only on the hormones present in the circulation but on the specific receptors and signal components expressed by the cell at any time. In effect, the pattern of different receptors carried defines cell type (e.g. a liver cell or an epithelial cell).
RAS-MAPK signalling has been extensively studied in the PC12 cell line, derived originally from an adrenal tumour. These cells proliferate in response to EGF but they are a useful model for neuronal differentiation because treatment with nerve growth factor (NGF) halts their proliferation and induces the formation of sympathetic neurons. Both responses are signalled via RAS-MAPK but with different requirements from two members of the RAF family, RAF1 and BRAF (ARAF is the third family member). Transient activation of RAF1 and BRAF promotes proliferation and it is only when BRAF activation is sustained that differentiation occurs (Fig. 3).
3. Sustained versus transient ERK activation: role of RAF proteins.
These cells have been subjected to numerous manipulations of receptors and signalling proteins with the consistent result that transient signalling leads to proliferation, whereas sustained BRAF activation promotes differentiation. Both receptors (EGFR and TRKA) are RTKs, contrasting signals being generated through subtle differences in the activated coupling proteins.
The tissue dependence of signalling can be illustrated by considering two types of a tumour. In the lung, tumour development is driven by RAF1 and BRAF can be dispensed with, at least in mice. However, in melanocytes (pigmented skin cells, from which melanoma develops) NRAS activates BRAF and MAPKK.
The underlying point is that, in principle, a mutation in any component of an intracellular signalling pathway that results in abnormal activity has the potential to convert a normal cell into one with the capacity to become cancerous.