Gene expression is regulated at the level of transcription In response to a wide variety of extracellular stimuli. During such a response, external signals have to be transmitted from the cell surface to the nucleus, a process known as signal transduction. Until recently only one signalling pathway was understood In detail from cell surface receptor to transcription factor I.e. the stimulation of CREB activity as described In section 1.2.2. However, biochemical studies In mammalian cells have delineated a second pathway, the mitogen activated protein kinase (MAPK) pathway.
1.4.1 The MARK Pathway
The MARK pathway Is stimulated by growth factor binding to the extracellular domain of receptor tyrosine kinases (RTKs). This Is followed by receptor dimerization, stimulation of protein tyrosine kinase activity and autophosphorylation (Ullrich and Schlesslnger, 1990). The phosphotyroslne and neighbouring amino acids function as a binding site for the Src homology domain, SH2, of adaptor molecules such as Grb2 which In turn recruit the guanlne- nucleotlde exchange factor, Sos, to the cytoplasmic surface of the plasma membrane (McCormick, 1993). There Sos catalyzes the dissociation of GDP from Ras so facilitating GTP binding (Aronhelm et al., 1994). This converts Ras Into Its active form and allows It to Interact with the next component of the pathway, Raf, thereby recruiting It to the plasma membrane. Membrane association of Raf, a MAPK kinase kinase (MAPKKK/MEKK), activates Its serlne/threonlne kinase activity (Leavers et al., 1994) and results In phosphorylation and activation of the next component In the pathway, MAPK kinase (MAPKK/MEK) (Kyrlakis et al., 1992). MAPKK Is dual specificity kinase which phosphorylates MAPKs on both tyrosine and threonine residues (Naklelny et al., 1992). This dual phosphorylation Is required to activate the MAPKs which then phosphorylate substrates at the minimal consensus sequence serlne/threonlne-prollne (Davis, 1993).
Thus, the MAPK pathway can be seen to consist of a kinase cascade triggered by Ras. The pathway can be stimulated In ways other than the RTK scheme described above e.g. certain G protein coupled receptors are able to stimulate Ras and Raf activity through the action of their effectors (reviewed In Blumer and Johnson, 1994). This extra Input of signals allows stimulation of the MAPKs In response to a wider variety of external stimuli.
1.4.2 MAP Kinase Homoiogs
Cloning of the MAPKs has led to the Identification of two subfamilies. These are the extracellular regulated kinases e.g. E rk i, 2 and 3 (Boulton et al., 1991) and the stress activated protein kinases, SAPKa, p and y (SAPKy Is also referred to as JNK1) (Derljard et al., 1994; Kyrlakis et al., 1994). The two families are 40-45% Identical In sequence and both require dual phosphorylation on key regulatory tyrosine and threonine residues In order to be activated. Furthermore, both families are activated by Ras. There are, however, distinct
differences which suggest that despite the similarities the families belong to separate signalling pathways.
Firstly, known MAPK stimuli differentially activate the two families. Mitogens such as epidermal growth factor (EGF), fibroblast growth factor (FGF) or phorbol ester strongly activate the Erks but have only a weak stimulatory effect on the SAPKs. Conversely, agents which induce cellular stress such as hydrogen peroxide, UV light and heat shock strongly activate the SAPKs but only weakly or moderately activate the Erks. Secondly, the MAPKK responsible for activating these proteins is different in the two cases; MEK1 and MEK2 phosphorylate and activate the Erks (Zheng and Guan, 1993) whereas SEK1 activates the SAPKs (Sanchez et al., 1994). Thirdly, the motif phosphorylated by the relevant MAPKK differs in sequence in the two groups; in the Erks it is Thr-Glu-Tyr but in the SAPKs it is Thr- Pro-Tyr. Finally, the two families differ in their substrate specificity e.g. the SAPKs phosphorylate GST-cJun fusion proteins containing the N-terminal activation domain of cJun much more efficiently than they phosphorylate myelin basic protein (MBP) whereas the opposite is true for the Erks.
A further MAPK homolog has been identified, p38/RK/p40 (Freshney et al., 1994; Han et al., 1994; Rouse et al., 1994), which appears to be distinct from the Erks since it is not activated by EGF or MEK1 and it does not phosphorylate MBP. Instead it is activated by heat shock which stimulates the SAPKs. p38, however, is distinguished from the SAPKs because it is unable to phosphorylate GST-cJun and its sequence of regulatory phosphorylation by MAPKK is Thr-Gly-Tyr (in the SAPKs it is Thr-Pro-Tyr). Therefore, p38/RK/p40 appears to be a novel MAPK and may thus represent the first identified member of a third MAPK subfamily.
1.4.3 Several MARK Pathways In Mammalian Cells
The identification of three potential MAPK subfamilies, two of which (Erks by MEK1 and 2; SAPKs by SEK1) are activated by different upstream MAPKKs, suggests that different but related MAPK signalling pathways exist in mammalian cells. This is further suggested by the identification of three distinct mammalian MAPKKKs: Raf-1 (Kyriakis et al., 1992), MEKK1 (Lange-Carter et al., 1993) and Mos (Nebreda et al., 1993). Two of these have recently been shown to stimulate independent pathways in vivo; Raf-1 activates MEK1 thus leading to stimulation of Erk activity whereas MEKK1 activates SEK1 thereby stimulating SAPK activity (Yan et al., 1994). The pathway leading to p38 stimulation has not yet been established. However, since the upstream activator of p38 is not MEK1, it suggests that p38 activation also occurs via a distinct signalling pathway.
Further support for the existence of independent mammalian MAPK cascades is given by the fact that three distinct MAPK-like cascades have been identified in yeast. One of these is involved in coordinating the physiological changes involved in mating, another is involved in cell wall construction and integrity, and the third is involved in adaptation to osmotic stress (reviewed in Ammerer, 1994). Erki and 2 are mammalian homologs of the yeast proteins
FUS3/KSS1 which lie on the mating pathway whereas p38 is a homolog of H0G1 which lies on the osmoregulation pathway (Rouse et al., 1994). This suggests that the different mammalian MAPK regulatory cascades have different physiological functions which is in agreement with the observation that the different MAPK subfamilies are generally activated by different stimuli and phosphorylate different substrates. However, there is also a certain degree of cross talk between the different pathways e.g. Ras is required for both Erk and SAPK activation, although Ras independent activation of the SAPKs is known. Furthermore, in certain cases the same stimuli can activate more than one pathway although to differing extents e.g. EGF strongly stimulates the Erks and also weakly activates the SAPKs. Only when all the components of the different signalling pathways have been identified can the true extent of overlap and interplay between the various cascades be determined.