Toma de decisiones

PtdIns(3)P

^ PtdIns(3,4)I^ ^ = ^ PtdIns(3,4,5)I^

J /

y /

/ V

Ptdlns ^

PtdIns(4)P

PtdIns(4,5)P,

Figure 1:5. Generation of 3-OH phosphoinositides. Shown here are the lipid products synthesised by the 3-phosphoinositide pathway. The solid

arrows indicate pathways which are known to occur in vivo. The dashed arrows

indicate activities that can be detected in cell lysates or with purified enzymes.

The role of PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 as second m essengers in signal transduction is an area o f intense discussion and controversy. These lipids are generated through the activation o f the Ptdlns 3-

kinase family. Certainly evidence exists for accumulation of PtdIns(3,4,5)P3 in

vivo in response to mitogenic stimuli (Stephens et al., 1991). The targets of such a second m essenger are not clear although in vitro evidence for the activation of some PKC isotypes has been presented (Singh et al., 1993; Toker et al., 1994; Nakanishi et al., 1993). More recently Rac (related to A and C

kinase; also known as Akt and PKB) - a serine/threonine kinase (Jones et al., 1995; Coffer and W oodget, 1995) - has been identified as a Ptdlns 3-kinase effector which is activated by PtdIns(3)P. These results have lead to Rac being placed downstream of Ptdlns 3-kinase in the wortmannin sensitive p7056kmase activation pathway (Franke et al., 1995; Burgering and Coffer, 1995).

The Ptdlns 3-kinase family catalyses the addition of phosphate to the D- 3 position of the inositol ring. In vitro Ptdlns3-kinases are able to use Ptdlns,

PtdIns(4)P as well as PtdIns(4,5)P2 as substrates, in vivo however mitogenic stimuli result in PtdIns(3,4,5)P3 as the major product indicating that PtdIns(4,5)P2 is the major substrate in the cell (Stephens et a l, 1991). The first described Ptdlns 3-kinase exists as a heterodimer comprised of two subunits, a p85 regulatory subunit which serves as a regulatory adaptor molecule and the catalytic p i 10 subunit (Figure 1:6). The p i 10 subunit of Ptdlns 3-kinase is homologous to the S.cerevisiae protein Vps34, a protein involved in vacuolar protein sorting (Schu et al., 1993) and which has been shown to contain intrinsic Ptdlns 3-kinase activity (Stack and Emr, 1994; Kodaki et al., 1994a). The p i 10 subunit of the Ptdlns 3-kinase contains not only lipid kinase activity but also an intrinsic protein serine threonine kinase activity and as such is a dual specificity kinase (Dhand et al., 1994; Woscholski et al., 1994). The Ptdlns 3- kinase family is one which is rapidly expanding - several p85 and p i 10 subunits having been identified to date (Stoyanov et al., 1995). The Ptdlns 3-kinase (p85/110 types) associates with and is activated by various growth factor receptors, i.e. PDGF-p, CSF-1, HGF and KIT as well as non-receptor tyrosine phosphorylated proteins such as IRS-1. In addition to the tyrosine kinase receptor coupled Ptdlns3-kinases an additional family member which is stimulated by G protein py subunits has been described (Stephens et al., 1994; Thomason et al., 1994). This novel Ptdlns 3-kinase, pllOy, has recently been cloned and is activated in vitro by both a and Py subunits (Stoyanov et al.,

1995). Further, this pi lOy isoform contains a potential PH domain and does not interact with a p85 regulatory subunit and as such has been proposed to link signalling through G protein coupled receptors to generation of phosphoinositides second messengers phosphorylated in the D3 position (Stoyanov et al., 1995).

BH3H H S H 2

p85

pllO

Figure 1:6. Schematic diagram of the Ptdlns 3-kinase family. The originally identified Ptdlns 3-kinase consists of a p85 adaptor subunit (p 8 5 a or p8SP) and a catalytic p i 10 subunit (pi 10a or p i lOp). Recently a py activated p i lOy has been described (see text) w hich does not bind p85. The p i 10 subunits share homology with the catalytic domain o f Vps34p, and contains sites for p85 and Ras binding in the amino-terminus. The p85 subunit binds to p i 10 via the in ter-S H 2 dom ain, a region w hich also co n tain s the phosphorylation site for the p i 10 protein kinase on p85. The p85 subunit also contains proline-rich regions (?) as well a Bcr homology region.

The elucidation o f signal transduction pathw ays involving Ptdlns 3- kinase have been aided by the discovery o f the antifungal com pounds wortmannin (WMN) and dem ethoxyviridin (DMV) which are potent inhibitors (low nanomolar) targeting Ptdlns 3-kinase both in vitro and in vivo (Kanai et

al., 1993; A rcaro and W ym ann, 1993; W oscholski et al., 1994). Such

com pounds have been em ployed in studies im plicating Ptdlns 3-kinase in the regulation of the cytoskeleton (Kotani et al., 1994; W ymann and Arcaro, 1994), in neutrophil activation (Arcaro and W ymann, 1993) as well as the binding and activation of Ptdlns3-kinase by the small G protein ras (Kodaki et al., 1994b; Rodriguez-V iciana et al., 1994). The accum ulating evidence highlights the im portant role for this family of lipid kinases and the phospholipids they produce in signal transduction processes within the cell.

1:8 The phospholipase families.

Several phospholipases are responsible for the hydrolysis of phospholipids in. the production of signalling molecules, these include phospholipase A2, sphingomyelinase, phospholipase D and phospholipase C.

Ptdlns-PLCs are responsible for agonist stimulated PtdIns(4,5)P2 hydrolysis, and PLC isoforms have been cloned from a number of sources (reviewed in Meldrum et ah, 1991). These enzymes range between 55 and 155 kDa can be subdivided into three major classes: PLCp, y and Ô (Figure 1:7) and show a generally conserved structural make-up, consisting of an X and Y domain and an amino terminal PH domain (see Section 1:4:3). Deletion of either or both of these domains, or introduction of even a particular point mutation into domain X of PLCy, inactivates the enzyme, suggesting that the conserved domains encode the catalytic activity (Bristol et al., 1988; Meldrum et al., 1991). In addition to an X and Y domain the members of the PLCy class contain two SH2 domains and an SH3 domain as well as a split PH domain.

Different classes of ligand receptor activate different classes of PLC. The serpentine receptors activate the PLCp class through G proteins and the RTKs activate the PLCy class by complex formation and tyrosine phosphorylation. For example, phosphorylation and activation of PLCy by a T- cell receptor (TCR) associated tyrosine kinase is responsible for TCR stimulated PtdIns(4,5)P2 hydrolysis (Cockroft and Thomas, 1992).

Many mitogens and growth factors result in a biphasic production of diacylglycerol (Leach et al., 1991). This was initially noted in the response of fibroblasts to a-thrombin (Wright et al., 1988). An initial rapid production of diacylglycerol is coincident with the production of inositol phosphates unlike the second sustained phase of production. Further analysis of the biphasic diacylglycerol response supported production of the initial phase from Ptdlns hydrolysis and the second through hydrolysis of PtdCh (Pessin et al., 1989). Data from bombesin treated Swiss3T3 cells has shown that the sustained

diaclyglycerol production (up to 30 minutes) observed in this case is due to the action of phospholipase D (PLD), as the headgroup released was choline and not choline phosphate (Cook and Wakelam, 1989).

PLC-p