PLANIFICACIÓN ESTRATÉGICA

Mitogen-activated protein (Map) kinase cascades are important intermediates in signal transduction pathways. Originally identified as mediators of mitogenic growth factors, it is now clear that Map kinase signalling cascades regulate cellular responses to a wide range of extracellular stimuli. Moreover, its is becoming evident that each Map kinase pathway has 'its own' GTPase as a molecular gatekeeper between input signal derived from the plasma membrane and the occurrence of the first phosphorylation events that eventually will activate the cellular genetic program (Fig. 1.9). An additional level of complexity derives from the notion that each of these GTPases activates more than one effector cascade (Marshall, 1996; Tapon and Hall, 1997).

1.5.1 The ras superfamily of small GTP-blnding proteins

The ras superfamily of GTP-binding proteins are all GTPases and function as molecular switches, cycling between an active GTP-bound and an inactive GDP-bound form. Over 50 members of the ras superfamily are currently known and based on their sequence homologies they have been grouped into seven subfamilies: ras, rho, rab, ARF, s a r i, ran and rad/gem. Members of the ras family include H-, Ki-, N-Ras; R-Ras; R a p i, 2; Ral; and TC21. Rho family members are RhoA, B, C; R a d , 2; Cdc42; RhoG; and TC10. Ras family GTPases are predominantly involved in processes leading to growth and differentiation. The biological functions of rho family proteins comprise the regulation of the actin cytoskeleton (see section 1.7.4), however more recently these GTPases have also been shown to be involved in signalling pathways leading to gene

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transcription (see below). The Rab, ARF, Sar1 and Ran GTPases play a role in vesicle transport and nuclear protein import, respectively.

GTPases are regulated by GTPase activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs). Increasing the activity of a GAP protein leads to inactivation, whereas increasing the GEF activity results in stimulation. The rho family of GTPases, in addition, can be regulated by GDP dissociation inhibitors (GDIs) which keep the GTPase in a locked GDP-bound form; upon activation the GDI is released which allows exchange of GDP to GTP. In T cells, Ras is activated by the TCR and the IL-2R but not by CD28 (when a physiological stimulus is used) (Downward et al.,

1990; Graves et al., 1992; Nunes et al., 1994). Its regulation by GAPs and GEFs in TCR and IL-2 signalling has been mentioned in the previous section and will be further discussed in chapter 3. The regulation of Cdc42, Rac and Rho proteins by surface receptors in T cells has not been addressed. However, Rac may be regulated by Ras or Vav (see below and chapter 3).

It is now recognised that Ras can activate multiple effector pathways mediated by specific interactions of Ras in its GTP-bound form with distinct downstream signalling molecules (Marshall, 1996). In this respect, mutants of Ras which selectively interact with only one effector have now been identified (Joneson et al., 1996; White et al.,

1995). Thus, Ras can interact with Raf and activate the Mek/Erk Map kinase pathways (see below). Studies on the role of Ras in fibroblast transformation have concluded that the Raf/Mek pathway does not mediate all Ras effector functions (White et al., 1995). Similarly, in T lymphocytes, the Raf/Mek pathway has been shown to mediate Ras effects on positive selection of thymocytes but apparently is not required for proliferation (Alberola-lla et al., 1995). Other molecules that could be involved in mediating the tremendous array of Ras responses include rasGAP (Downward, 1992), PI 3-kinase (Rodriguez-Viciana et al., 1994) and Ral-GDS (GEF for Ral) (Hofer et al., 1994).

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Moreover, Ras and Rho are essential for Ras transformation (Khosravi-Far et a!., 1995; Qiu et a!., 1995; Qiu et a!., 1995) and rho family proteins may control Ras effects on the actin cytoskeleton (Ridley et al., 1992). In T cells, Rac may mediate some of the functions of Ras leading to induction of the transcription factor NF-AT (see below).

Similar to Ras, it has now been shown that the rho family proteins Cdc42, Rac and Rho activate at least two effector pathways, one leading to gene transcription and one leading to rearrangement of the actin cytoskeleton (see below, section 1.7.4 and

chapter 6). Moreover, these GTPases seem to be required for the progression through

G1 of the cell cycle (Olson et al., 1995). These distinct functions are thought to be reflected by the interaction of these GTPases with different effector molecules (Lamarche et al., 1996; Tapon and Hall, 1997; Westwick et al., 1997).

1.5.2 Map kinase modules

Map kinases (MAPK, see Fig. 1.9) are proline-directed kinases which phosphorylate sites containing the core consensus motif Ser/Thr-Pro. Such motifs are found in the transactivation domains of a wide range of transcription factors and one feature of Map kinases is their ability to translocate into the nucleus where they can directly phosphorylate these sites (Fig. 1.9). Alternatively, they can also phosphorylate and hence regulate additional protein kinases such as Mapkapk2 (Cuenda et al., 1995; Rouse et al., 1994; Stokoe et al., 1992) or Rsk (Blenis, 1993). Three mammalian Map kinase groups have been characterised - the Erks (extracellular-signal regulated kinase), the Jnks/Sapks (c-Jun N-terminal kinase / stress-activated protein kinase) and the p38/RK/Mpk2 (kinase homologous to Hogi of S. cerevisiae] RK, Reactivating kinase) - a fourth group may exist in one that is regulated by the GTPase Rho and impinges on serum response factor (SRF)-mediated gene transcription (Fig. 1.9); reviewed in (Cahill et al., 1996; Cano and Mahadevan, 1995; Davis, 1994; Marais and Marshall, 1996; Marshall, 1994; Treisman, 1996; Woodgett et al., 1996). Map kinases themselves are