Etapas del TPM

Wortmanin, a fungal metabolite, and LY294002, a bioflavoid derived from quercitin, are low molecular weight, cell permeable inhibitors of PI3K activity (Vanhaesebroeck et at., 1997). Wortmanin binds irreversibly to Lys608 on the p i 10 subunit of PI3K by forming a covalent bond, and inhibiting this PI3K with an IC5 0 in the low nanomolar

range (l-5nM) (Arcaro and Wymann, 1993; Thelen et al., 1994; Woscholski et al.,

1994; Yano et al., 1993). Woitmannin has been used to identify downstream PI3K signalling pathways which are dependent upon PI3K catalytic activity (Okada et al.,

1994; Thelen et al., 1994; Yano et al., 1993). Woitmannin does not however inhibit all classes of PI3K to the same extent. Although woitmannin inhibits all known class I PI3Ks and human Vps34 with a similar IC5 0 (Stephens et al., 1994; Stephens et al.,

1994) yeast Vps34p, a bovine homologue and PI3KC2a have lower sensitivity

The main limitation to the use of PI3K inhibitors in the investigation of cellular physiology is their lack of specificity. They fail to distinguish between different isoforms of PI3K and are known to also inhibit some isoforms of Ptdlns 4-Ks, TOR kinases and other signalling molecules such as cytosolic phospholipase A2.(Hartley et al., 1995; Yano et al., 1995). Since the concentration of the inhibitor inside the cell cannot be determined with confidence, the specificity of PI3K inhibitor effects should be treated with caution.

1.4.7.8 Upstream regulation of PI3K

Class I PI3K have been demonstrated to play a role in a large range of receptor mediated signalling events (Figure 1.24) (Cantley etal., 1991; Stephens et al., 1993; V articovski et al., 1994). When receptor stimulation by a ligand results in phosphorylation upon a tyrosine residue within a YXXM motif, phosphotyrosine residuesserve as a docking site for the SH2 domains of the adaptor subunit of class I PI3Ks (Kapeller and Cantley, 1994). Such adaptor-mediated translocation brings the catalytic subunit into close proximity with its lipid substrates in the cell membrane. Binding of a phosphopeptide which mimics the p85 SH2 domain binding site on activated receptors has also been reported to directly increase the activity of the catalytic subunit by two to four fold (Rordorf Nikolic et al., 1995).

The paradigm for receptor mediated activation of PI3K is the PDGFp-R. PI3K was first shown to be important in PDGF stimulated mitogenesis in studies through the use of PDGF|3-R mutants in which tyrosine residues that were targets of the p85 SH2 domains were mutated to phenylalanine. When transfected into human Hep G2 cells, these mutant receptors were found to be defective in their ability both to recruit PI3- kinase and to respond to PDGF as a mitogen (Valius and Kazlauskas, 1993). Subsequently, microinjection of neutralising antibodies specific for the p i 10a subunit has been shown to block the growth stimulatory effect of PDGF and EGF in quiescent fibroblasts (Roche et al., 1994). PDGFpR mutants lacking the PI3K binding site have also been used to demonstrate the importance of PI3K activity for receptor internalization and degradation (Joly etal., 1995).

An alternative mechanism by which cytosolic PTKs may recruit and activate PI3K involves SH3 domain interactions. SH3 domains from the src family have been shown to associate with the proline-rich motifs of p85a which results in the activation of PI3K activity (discussed in Chapter 5). The small GTP-binding protein Cdc42, has been shown to interact with the BH domain and activate the lipid kinase (discussed in Chapter 4), but the physiological significance of both these results is yet to be established.

Class I PI3Ks have also been shown to interact with Ras proteins in a GTP-dependent manner. This interaction has only been studied in detail for the pi 10a/p85a complex (Marte etal., 1997; Rodriguez Vicianaef a/., 1994; Rodriguez Vicianagf a/., 1996). The Ras-related proteins Rac, which has been implicated in signalling downstream of PI3Ks (Hawkins et al., 1995), and Rho, do not bind pllO a/p85a (Rodriguez Viciana

et al., 1994). RasGTP bound an amino terminal region of the p i 10 subunit and activated PI3K invitro. Transfection of activated PI3K and an activated Ras mutant, Val 12 Ras into PC12 cells resulted in elevated levels of Ptdlns 3,4-P2 and PtdIns(3,4,5)P3. Dominant-negative ras (type N17, in which Serl7 is mutated to Asn) blocked the increase in levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in response to EGF and nerve growth factor (NGF) stimulation. Accumulation of GTP-bound Ras has been shown to be required for full activation of classlA PI3Ks by PDGF (Klinghoffer et al., 1996). It therefore appears that PI3K activation is downstream of Ras activation, however, it is not yet known whether PI3K activation by RTK’s also occurs independently of Ras

PI3Ks may thus be another class of Ras effector molecules, together with proteins such as Raf Ser/Thr kinases (section 1.3.4.1.3). The interaction of Ras with a PI3K may

also result in allosteric activation of PI3K and /or contribute to PI 3K recruitment to the plasma membrane. Interestingly, Ras effector mutants have been identified that interact with p i 10a but not with Raf-1 and vice versa (Kauffmann Zeh et al., 1997; Rodriguez Viciana et al., 1997). suggesting that PI3K is part of a Raf-independent signalling pathway downstream of Ras. Conversely, it has been reported that expression of a constitutively active form of PI3K results in induction of Fos transcription, which is blocked by both N17 dominant-negative Ras and a dominant-negative form of Raf (Hu

et al., 1995). Furthermore, GTP-bound Ras is elevated in cells transfected with a constitutively active form of PI3K, which suggests that Ras is downstream of PI3K. This hypothesis is supported by experiments in which microinjection of the amino- terminal SH2 domain of p85a as a dominant-negative inhibitor of PI3K blocks induction of Fos transcription in response to insulin stimulation (Jhun et al., 1994). This block was rescued by expression of an activated form of Ras. Inhibition of Fos induction by a neutralising antibody raised to the amino terminal SH2 domain of p85a was also rescued by expression of activated Ras.

At present it is unclear how heterotrimeric G-proteins activate the class IB PI3K. Whilst Gpy can directly stimulate pllOy lipid kinase activity this stimulation is considerably enhanced in the presence of the plOl adaptor (Stephens et al., 1997). The regulation of the class II PI3K activity remains unknown. Similarly for the class III PI3Ks, it can be hypothesised that the class II PI3Ks may be constitutively active (based on the housekeeping role of the yeast Vps34 in vesicular trafficking).