Ras regulates multiple effector pathways which in turn control the

Ras signals regulate Elk-1 in a variety of systems. There is diversity at the level o f the kinase which directly acts upon Elk-1, depending on the nature o f the initiating signal input. Elk-1 regulation by different MAP kinases could reflect 1) that m ultiple parallel GTPase/kinase cascades (eg derived from Ras and terminating in ERK, derived from Rac and terminating in INK) can regulate Elk-1 or 2) that Elk-1 is a target for different effector pathways which diverge downstream o f Ras itself.

There is now much evidence that Ras and other GTPases regulate multiple signalling pathways. Molecules that interact with Ras.GTP (Ras effectors) and GTP bound form s of other GTPases are being identified in increasing num bers, by techniques varying from yeast-two-hybrid screening to direct 'pull-down' assays of G TP d ependent interactions. M oreover, analysis o f the G TP d ependent conform ational changes in Ras molecules allowed rational deign o f mutants with altered effector selectivity. Two regions of Ras alter their conformation in a nucleotide dependent manner. The switch II region is N-terminal and highly mobile. The Switch I region is composed of residues 30-40, point mutational analyses of this region have shown that certain mutations ablate the ability of oncogenic (Val 12) Ras to signal but do not affect GTP binding (Marshall, 1996). These effector mutations are altering key residues that are the specificity determinants of Ras.GTP interaction with a particular effector molecule. Ras interacts with diverse targets in evolutionarily divergent organisms. Genetic data from yeast S. pomhe and S. cerevisiae indicate that there are multiple targets for Ras in each organism (Chang et aL, 1994). There is also evidence for multiple Ras effectors in higher organisms (White et aL, 1995).

Characterised Ras effectors are shown in Figure 4.3 and reviewed more fully in (Marshall, 1996). These are proteins which have been demonstrated to interact with Ras.GTP. The relative importance or even existence o f these interactions across a spectrum of cell types is not well understood for all effectors. The best characterised case is interaction with Raf-1 and subsequent induction of the M EK/ERK signalling cascade. In addtion to the direct effector protein interactions shown below, there is a class o f indirect transducers of Ras.GTP dependent signals. For example, there is considerable evidence that Ras family GTPases may act in cascades. Thus Rac-1 and Rho can act downstream of Ras in regulation of growth factor induced rearrangements

o f the fibroblast actin cytoskeleton. It is also established that oncogenic transformation by Ras can be Rac-1 dependent and that by Rac-1 can be Rho dependent.

u

1

EFFECTOR

Raf-1

Ral-GDS

NF-1

pl20 GAP

PI 3-kinase

Rac

MEKK-1

ACTIVITY

Ser/Thr kinase. Directs induction of the MEK/ERK cascade.

Guanine nucleotide dissociation inhibitor of the Ral GTPase. Promotes GTP/GDP exchange. Role in oncogenic transformation

by Ras.

Neurofibromin-1. GTPase activating protein for Ras

GTPase activating protein for Ras

Phosphatidy inositol 3'hydroxyl kinase. Possible roles in directing Ras and Rac mediated actin cytoskeleton rearrangement.

Rho family GTPase. Rac-1 may transduce Ras signals to the actin cytoskeleton or towards

oncogenic trcinsformation

MAP KKK (ie. Raf-1 equivalent) in the signaling cascade terminating in Jun kinase

activation.

F ig u r e 4 .3 . R a s e ffe c to r s id e n tifie d in m a m m a lia n c e lls . T h e s e p r o te in s p r o v id e potential for a single upstream input to Ras to regulate diverse cellular outcom es. For references see (Marshall, 1996) and citations therein.

4 .1 .4 Antigen receptors induce immediate early genes.

The regulation of the c-fos gene by the F ceR l is established (Lewin et aL,

1996; Stephan et aL, 1997). The FceRl is also thought to induce c-jun activity, but regulation o f any of the numerous other immediate early genes has not been examined in this system. The protein product of the c-fos proto-oncogene, Fos, is an im portant transcription factor in leukocytes. It is a partner in the AP-1 transcription factor

com plex which is known to participate in the regulation o f a variety o f im m une response genes. AP-1 homo- or hetero-dimers are required for the optimal activity of N FA T (N uclear Factor of A ctivated T cells). NFAT/AP-1 transcription factor complexes control induction of multiple immune response genes including those for IL-2, IL-4, IL-3, GM-CSF and the Fas- and CD40-ligand m olecules (Rao et aL,

1997). The F ceR l regulation of NFAT is a major topic o f this thesis and will be returned to in detail later.

Elk-1 is highly expressed in B- and T- lymphocytes (Bassuk and Leiden, 1997) Regulation of E lk -1 transcriptional activity by the T cell antigen receptor has been described (Genot et aL, 1996). W hilst the Elk-1 regulation o f the c-fos prom oter is well established, no hematopoietic specific gene targets for Elk-1 have yet been identified. The co-operative role o f Fos and NFAT family m em bers does how ever suggest that regulation of Elk-1 may be an important consequence o f antigen receptor stimulation. Moreover, Elk-1 may well have non-SRE sites o f action in promoters other than that of the c-fos proto-oncogene. Taken together the importance of Elk-1 as a gene regulator in many systems and its clear regulation by GTPase/kinase cascades suggest that this may be an interesting object of study in the RBL2H3 system. In order to look at Elk-1, a transient transfection approach was em ployed using a CAT (Chloramphenicol acetyl transferase) reporter gene system established by the laboratory of Dr Richard Treisman at ICRF (Marais et aL, 1993).

E lk -1 is clearly a target for Ras in fibroblasts. There has previously been no examination of targets for Ras mediated signals in RBL2H3 or o f the regulation o f immediate early gene induction. The identification of Ras.GTP targets in the F ceR l regulated RBL2H3 is an objective of the work presented in this thesis. Therefore the following questions were asked:

1) Is Elk-1 a target for Ras family GTPase signals in FceR l stimulated RBL2H3? 2) Is Elk-1 regulated by a specific MAPK Ser/Thr kinase cascade in Rbl2H3?

4 .2

RESULTS

4 .3

The LexAOPtk.CAT/LexA Elk-1 reporter gene system for