Fos, from feline osteosarcoma virus.

Myc, the cellular counterpart of the transforming gene of the avian leukosis retrovirus MC29.

Jun, from avian sarcoma virus-17: we are informed that ju-nana is 17 in Japanese.

Note: abbreviations for genes are presented in lower case: fos, myc, etc. The same abbreviations with the first letter capitalized indicates their respective gene products (proteins).

Ets and Elk. Ets (short for E-26 specific), comprises a family of transcription factors. Ets was discovered as a transforming factor, v-ets, from acute avian leukaemia retrovirus E26. This virus causes erythroblastosis mouse and myeloblastosis in chicken.131 Thereafter, numerous mammalian

homologues, c-ets, came to light.132 One of them is ELK-1, Ets-like factor-1, which was first identified as part of a complex

of three components together with p67SRF and DNA. It was therefore initially referred to as ternary complex factor or p62TCF (see page 424).

through this attachment it encounters eIF-4E. How phosphorylation of eIF-4E facilitates initiation of protein synthesis is not clear.122,123

All in all, the stimulatory role of the MNKs on the initiation of protein synthesis is somewhat modest. Far more significant is phosphorylation through the

PI 3-kinase/PKB branch of the rPTK signal transduction pathway, discussed in Chapter 18.

Interestingly, MNK1 is the target of some of the viruses that hijack the protein synthetic apparatus. The p100 gene product of adenovirus binds eIF-4G and displaces MNK1 so that it is no longer able to phosphorylate and activate eIF4E. As a result, the cellular mRNA remains untranslated, but the translation of viral mRNA is unaffected. The cell becomes a machine for the manufacture of viral proteins.124

Activation of early response genes

The early response genes become activated within 20 min of receptor stimulation. Their activation is transient and can occur under conditions in which protein synthesis is inhibited. Activation of the EGF receptor causes rapid induction of c-fos, one of the first cytokine-inducible transcription factors to be discovered.125 It occupies a central position in the regulation of gene expression. Other early response genes include c-myc and c-jun.

As a result of the double phosphorylation by MEK, ERKs 1 and 2 undergo dimerization and a proportion translocates into the nucleus. How this occurs is not clear. Not only does ERK itself not possess nuclear localization signal (NLS), but it is far from clear whether the proteins that bind ERK cause its nuclear localization. Both passive (diffusion) and active (Ran-GTP-mediated) transport have been demonstrated.126 It is possible that the ERKs actually localize to the nucleus by default, but are continually returned to the cytosol by their interaction with MEK, present in both compartments and the bearer of an unmistakable nuclear export signal.127

The promoter region of the c-fos gene contains a serum response element (SRE), a DNA domain that binds the transcription factors p67SRF (serum response factor) and Elk-1 (p62TCF). Phosphorylation of Elk-1 by ERKs 1 and 2 increases the formation of a complex of both transcription factors with the DNA to promote transcription of the c-fos gene128 (Figure 12.17). Sustained activation of ERKs 1 and 2 (and of the downstream kinase RSK1) also results in multiple phosphorylation of the newly synthesized c-fos that acts to increase its life time.129,130

The transcription factor c-Myc is yet another nuclear substrate of ERKs 1 and 2. It constitutes one of the master switches regulating protein synthesis and

hence cell proliferation. Together with its partner Max, it controls the activity of DNA-dependent RNA polymerases types I, II, and III. c-Myc is phosphorylated

Fig 12.17  Activation of transcription by ERK2. Within the nucleus, ERK2 phosphorylates Elk-1 which then associates with SRF to form an active transcription factor complex. This binds to DNA at the serum response element (SRE). ERK2 also phosphorylates and stabilizes c-Fos, which, in complex with c-Jun, binds to DNA at the AP-1 sequence. Both SRE and AP-1 induce strong expression of c-Fos as well as genes involved in the onset of cell proliferation (e.g. cyclin D).

by ERKs 1 and 2 within the transactivation domain and this stimulates its transcriptional activity.134,135 By regulating c-Myc activity, the Ras-MAP kinase pathway controls the quantity of ribosomes and thus protein synthesis.

Regulation of the cell cycle

Induction of cell proliferation requires that all the relevant genes are made accessible to transcription factors and all the enzymes needed for DNA replication expressed.137 A key starting point is the expression of cyclin

D, which, together with the kinase Cdk4, drives progression through the early (G1) phase of the cell cycle. Cyclin D1/Cdk4 renders DNA accessible to gene transcription by phosphorylating and inactivating retinoblastoma proteins (Rbs). Transcription of cyclin D1 is facilitated by ERKs 1 and 2 through induction of the transcription factors Fra1, Fra2, c-Jun, and Jun-B, all components of the AP-1 complex138 that, when phosphorylated,139 bind to the promoter of the cyclin D1 gene and drive its expression.140 The ERKs

are also instrumental in the formation of stable cyclinD1/Cdk4 complexes.141 Finally, ERKs 1 and 2 also phosphorylate and inactivate the transcriptional corepressor Tob1, that normally silences expression of cyclin D1.142

RNA polymerase I, present in the nucleolus, transcribes the 45S preribosomal RNA molecule (later contributing to the small and large ribosomal particles).

RNA polymerase II transcribes the ribosomal proteins ( 30 of them).

RNA polymerase III transcribes the 5S

ribosomal particle (which associates with the big ribosomal particle).136

The Rb protein was first identified as the product of a gene that is deleted in patients with retinoblastoma, a

tumour originating in the retina.143 The Rb protein is a member of the family of nuclear pocket-proteins that bind and inactivate the E2F transcription

factor and prevent cells from entering S phase. Inactivation of the Rb gene occurs in numerous other tumours. In the absence of Rb, cells enter into S phase more readily and they do not require the normal array of extracellular growth signals conveyed upon the expression and

activation of cyclinD/cdk4 in order to proceed.

Exactly how ERKs 1 and 2 contribute to the expression of the enzymes needed for DNA replication is not known. In the previous section we pointed out that translocation of ERKs 1 and 2 to the nucleus, signals the activation of immediate early genes, many of which are themselves transcription factors. These induce expression of a second wave of genes and eventually to the expression of DNA replication genes. Examples are those encoding helicases, topo-isomerases, DNA polymerases, and ligases. Beyond their roles in transcription, ERKs 1 and 2 also phosphorylate and activate carbamoyl phosphate synthetase II (CPS II), that catalyses the rate-limiting step in the de novo synthesis of pyrimidine nucleotides (dCTP and dTTP).144

In the case of the EGF receptor, the activation of ERK and its translocation into the nucleus is an absolute requirement for cell proliferation. Forced retention in the cytoplasm suffices to block growth-factor-induced DNA replication.145 Weak signals that fail to induce cell proliferation also cause ERKs 1 and 2

to move into the nucleus, but it then recycles to the cytosol within 15 min. A strong mitogenic signal can ensure nuclear retention for up to 6 h.146 Importantly, the signal must also be reinforced by signals from cell adhesion complexes.147 We return to the question of cooperation between growth factor receptors and cellular adhesion in Chapter 13 (see page 404).

Fine tuning the Ras-MAP kinase pathway: scaffold proteins

We have explained that a sequential series of protein modifications determines the propagation of cell-surface signals into the nucleus. A second tier of regulation is presented by the interaction of scaffold proteins with components of this signalling cascade. Scaffold proteins bring these signalling components together or hold them apart.

The names of yeast genes usually have three letters plus numbers and often reflect the phenotype of a mutant. Thus STE denotes sterile. By convention genes are uppercase/italic (STE5), mutant genes are lower case/italic (ste5) and gene products are simply capitalized, e.g. Ste5.

MAP kinase scaffold proteins discovered in yeast

The physiological relevance of scaffold proteins to MAP kinase activation was first demonstrated in Saccharomyces cerevisiae (baker’s yeast). Here, in the response to mating pheromones (ligands that interact with 7TM receptors, see page 113), Ste5 acts as a scaffold that binds and links Ste11

(a MAP3K), Ste7 (a MAP2K), and Fus3 (a MAPK) to form a MAP kinase signalling cassette.148,149 In addition, Ste5 also links the cassette to Ste4 and Ste18

(G protein -and - subunits, respectively) and to Ste20 (equivalent to p21activated kinase or PAK) (see Figure 12.18a).

Actually, Ste5 is more than a scaffold protein. It imposes conformational changes on components of the MAP kinase pathway which lead to kinase activation. For instance, it amplifies the signal by modifying the conformation of Ste11 following its phosphorylation by Ste20. It also

C-TAK1: Cdc25Cassociated kinase-1, not to be confused with TAK1, the acronym for TGF - activated kinase-1.

IMP is the abbreviation for ‘impedes mitogenic signal propagation’.

It is not inosine monophosphate!

as scaffold proteins (Figure 12.19). Although not strictly required, their presence greatly enhances signalling through this pathway.156

KSR1 is permanently fixed to MEK in a complex that also contains the serine/ threonine kinase C-TAK1 and two subunits of the phosphatase PP2A (PP2A- A (catalytic) and PP2A-C (regulatory). In quiescent cells the complex can be recovered from the cytosolic fraction, linked to another scaffold protein,

14-3-3 (Figure 12.19). In response to activation by growth factors, the complex containing KSR1 translocates to the cell membrane and promotes the formation of a signalling cassette.157,158

This translocation and subsequent assembly of the Ras–ERK signalling cassette involves detachment of 14-3-3 and then destruction of the inhibitor IMP. The first event is controlled by PP2A (Figure 12.19), the second by interaction of IMP with RasGTP (Figure 12.20). In the absence of growth factor signals, KSR1 is kept in a phosphorylated state by the kinase C-TAK1159 and the complex retained

in the cytosol through its binding to 14-3-3 (see Figure 12.19). On activation of growth factor receptors, PP2A dephosphorylates KSR1 allowing it to dissociate from 14-3-3, so leaving room for other binding partners at the membrane.