Cirugía Oral

(TAO). Both of these kinases act on MEK3 and MEK6 , which themselves

act on p38, which acts as the final effector protein of this pathway. TA Kl and TAO are also able to act on MEK4, which is a component in Jnk activation (as reviewed by Robinson and Cobb, 1997).

1.3.2.4. Transcription factors activated by MAPKs.

Once MAPK proteins have been activated by upstream signalling proteins they migrate to the nucleus and regulate the activity of transcription factors by phosphorylation of serine or threonine residues. Factors that have been identified as targets for MAPK phosphorylation include: E lk l, which has multiple phosphorylation sites acted upon by ER K l/2, and Jnk; Ets-1 and Ets-2, which are acted upon by ERK2; the related proteins Jun, which is acted upon by Jnk, and Eos, which is acted upon by ERK l/2; ATF-2 which is activated by both Jnk and p38. It is likely that many more substrates of MAPK proteins remain to be discovered (as reviewed by Treisman, 1996).

1.3.2.5. Effects of MAPK activation.

In all mammalian ER K l/2 cells are activated by mitogens, and prolonged translocation of ERKs to the nucleus increases expression of cyclin D l, thus inducing cells to enter the cell cycle. In fact a constitutively active ERK proteins lead to cell transformation. In some cell lines, ERK activation has been associated with differentiation. High levels of ERK proteins are expressed in all cells, and half of all ERK in activated cells is associated with the cytoskeleton, suggesting that ERKs have a role in cytoskeletal organisation.

Jnk has also been shown to be activated after some mitogenic stimuli, although this effect could also be due to an anti-apoptotic effect of Jnk activation in some cells. In contrast p38 activation is thought to inhibit cell proliferation, as p38 activation leads to an inhibition of cyclin D l

chanter 1 Introduction

expression. Jnk activation has been associated with hepatic regeneration and T-cell activation.

As their name implies, the stress MAPKs, Jnk and p38, are activated in response to stresses on the cell such as heat shock, osmotic shock, cytokines, antioxidants, UV light and DNA-damaging agents. The consequence of this activation appears to be growth arrest, and in some cases apoptosis, although it is thought that Jnk/p38 activation in isolation is not sufficient to induce apoptosis (as reviewed by Robinson and Cobb,

1997).

1.3.3. Phosphoinositide 3-kinase (PI3-K ).

PI3-K phosphorylates the D-3 position of the inositol ring of phosphatidylinositol (Ptdlns). This produces Ptdlns 3-P (where P = a

phosphate group), from Ptdlns; Ptdlns 3 ,4 -P2, from Ptdlns 4-P; and

Ptdlns 3,4,5- P3, from Ptdlns 4,5- P2. The most important of these

products is Ptdlns 3,4,5- P3. More than one form of PI3-K has been

identified, but the best defined kinase consists of a dimer of a p85 regulatory sub-unit, and a pl l O catalytic sub-unit. The p85 sub-unit consists of a single SH3 and two SH2 domains. The pl l O sub-unit consists of a p85 binding domain, a Ras binding domain, a lipid kinase unique domain, and a catalytic domain (reviewed by Carpenter and Cantley,

1996). Activation of PI3-K has been associated with FcyR-mediated phagocytosis (reviewed by Sanchez Mejorada and Rosales, 1998), as well as being central to TCR mediated signalling (Qian and Weiss, 1997).

1.3.3.1. Regulation of PI3-K activity.

The levels of the lipids that act as substrates for PI3-K are tightly regulated in cells. A further level of regulation is that the catalytic activity of PI3-K can be inactivated by phosphorylation of serine and threonine residues in the p85 regulatory domain. This protein kinase activity is

chapter 1 Introduction

domain. The SH2 and SH3 domains of the p85 sub-unit have been shown to interact with specific domains of receptor tyrosine kinases such as the receptors for platelet derived growth factor (PDGF) and epidermal growth factor (EGF) (as reviewed by Carpenter and Cantley, 1996).

1.3.3.2. Functions of PI3-K metabolites.

Studies using PI3-K inhibitors, such as wortmannin, dominant-negative forms of PI3-K and constitutively active forms of PI3-K, have revealed four distinct functions of PI3-K products. These four functions (outlined below), result from the interaction of the Ptdlns products of PI3-K with motifs such as Pleckstrin homology (PH) domains. These domains specifically bind phospholipids, and are important in activation of protein kinases, and in positioning of signalling proteins to the cell membrane,

PI3-K has been shown to play an important role in mitogenesis. Inhibition of catalysis by the p i 10 sub-unit blocked the DNA synthesis that is normally induced by PDGF and EGF. As described previously PI3-K, is able to interact with Ras-GTP via its p i 10 sub-unit. It has been shown that increased levels of activated Ras-GTP lead to an increased concentration of PI3-K-derived products, suggesting that PI3-K is downstream of Ras. However, addition of constitutively active PI3-K leads to an increased concentration of active Ras-GTP, suggesting that PI3-K is upstream of Ras. This implies that the Ras-PI3-K interaction is complex.

PI3-K activity has been shown to have an anti-apoptotic effect, which was demonstrated on serum starved PC 12 cells which spontaneously apoptose. This apoptosis is inhibited by transfection of constitutively active wild type PDGF receptor, but not by a mutant PDGF receptor that lacked a PI3-K binding site.