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The products of the lipid kinase activity of PI3K, PtdIns(3,4)P2 and PtdIns(3,4,5)P3

are thought to transmit PI3K signals by binding to other cellular proteins and modulating their activity or cellular location (Figure 1.25). Two modules present in many proteins, the PH domain (section 1.2.7) and the C2 domain (Newton, 1995) have been shown to specifically bind phosphoinositides, therefore PH and C2 domain containing proteins are the prime targets for effectors of PI3K transduced signals. It is unclear whether the protein ser/thr kinase activity of PI3K also results in signal propagation. Phosphorylation of p85 and pi 10 by PI3K results in a decrease in lipid kinase activity, and this may be part of a negative feedback loop. There is one report that IRSl can serve as a substrate for the ser/thr kinase activity of PI3K, but the significance of this phosphorylation event has yet to be determined.

1.4.7.9.1 PDK l-PK B/A kt

The Akt/PKB/RAC ser/thr kinase is the cellular homologue of the retroviral oncogene v-akt and is activated upon ligand stimulation of receptor tyrosine kinases (Bellacosa et

al.y 1991; Burgering and Coffer, 1995). A large number of studies which suggest that Akt is downstream of PI3K. Treatment of cells with the PI3K inhibitors Wortmannin or LY294002, or over-expression of dominant negative forms of PI3Ks inhibit Akt activation downstream of RTK stimulation (Burgering and Coffer, 1995; Franke et al.y

1995; Kohn etal.y 1996). In addition, overexpression of PDGPP receptor mutants that lack the PI3K binding site also fail to activate Akt (Burgering and Coffer, 1995; Franke et al.y 1995). Elevation of levels of PI3K lipid products in cells by overexpression of constitutively active mutants of p i 10a results in activation of Akt (Didichenko et al.y 1996; Klippel et al., 1996; Marte et al., 1997). Akt is a ser/thr kinase which contains an amino-terminal PH domain that has been shown to bind PI(3,4,5)Pg and PI(3,4)P2 with relatively high affinity and specificity (Franke et al.,

1997; Freeh et al., 1997; James et al., 1996; Klippel et al., 1997). Binding of PtdIns(3,4)P2, but not PtdIns(3,4,5)P3, to the PH domain of AKT increases its kinase

activity (Franke et a i, 1997; Freeh et al., 1997; Klippel et al., 1997). PtdIns(3,4)P2 is

mainly produced by the action of 5-phosphatases on PtdIns(3,4,5)P3 in vivo

(Woscholski and Parker, 1997), suggesting that 5-phosphatases act downstream of PI3K in signalling via Akt. Binding of PtdIns(3,4)P2 also induces the dimerization of

Akt in vitro (Franke et al., 1997). It is not clear whether dimerization directly affects kinase activity, although it has been shown that binding of a recombinant Akt kinase domain to Akt in vitro increases its kinase activity (Datta et al., 1995). Full activation of Akt requires two phosphorylation events as well as PtdIns(3,4)P2 binding. In

insulin or IGF-1 stimulated cells, Akt becomes phosphorylated on thr 308, which is in the kinase domain, and ser 473, which is in the carboxy-terminal tail, in a wortmannin sensitive manner (Alessi et al., 1996). In the presence of PtdIns(3,4,5)P3^ 3-

phosphoinositide-dependent protein kinase l(PDKl) phosphorylates Thr 308 and PDK2 phosphorylates Ser 473 (Alessi et al., 1997; Stephens et al., 1998; Stokoe et al.,

1997) Akt therefore integrates signals from several pathways, at least two of which involve the lipid products of PI3K. Akt can also be activated by stresses such as heat shock or hyper osmolarity in a PI3K independent manner (Konishi et al., 1996).

Activation of Akt mediates the insulin-induced inhibition of glycogen synthase kinase 3 (GSK3) which phosphorylates and down regulates glycogen synthase (Cross et at.,

1995), as well as eIF2B, an initiation factor important in the control of mRNA translation and protein synthesis (Welsh et al., 1996). A number of other regulatory proteins are also thought to he downstream of GSK3.

1.4.7.9.2 p7 0S6 kinase (p70S<»k)

p70^^ kinase(p70^^^) phosphorylates the 40S ribosomal protein S6 in response to

phase of the cell cycle (Brown and Schreiber, 1996; Kozma and Thomas, 1994; Proud, 1996). Activation of is regulated by multiple independent phosphorylations on serine and threonine residues. Activation of p70^^^, is not dependent on the Raf/MAPK pathway, but does involve PI3K and the PIK-related kinase, mTOR, as well as PKC and an unidentified proline-kinase (Brown and Schreiber, 1996; Kozma and Thomas, 1994; Proud, 1996).

Overexpression of receptor mutants which do not bind the p85 adaptor and PI3 kinase inhibitors both block ligand induced activation of p70^^ kinase (Chung et al., 1994). Furthermore, constitutive PI3K activity results in the phosphorylation of p7 0^^ kinase

and antisera which target p70^^ kinase and block the mitogenic activity of PI3-kinase (Weng et at., 1995).

Mammalian TOR (mTOR) is inhibited by the immunosuppressant drug, rapamycin, which is an analogue of another immunosuppresent, FK506. Rapamycin binds a 12kDa FK506 binding protein (FKBP12) which then binds to and inhibits mTOR. Rapamycin blocks the activation of p70^^^ by all known agonists, however it is also known that mTOR does not phosphorylate p7 0s6k directly. mTOR protein kinase

activity is also inhibited by Wortmannin, but PI3K is insensitive to rapamycin, thus the differential effects of these inhibitors have been used to the pathway involved in the regulation of p7Qs6K Inhibitors of PI3K such as overexpressed receptor mutants

lacking the PI3K binding site or wortmanin block p70S6K phosphorylation (Cheatham

et al., 1994; Chung et al., 1994) and expression of constitutively active forms of PI3K leads to increased phosphorylation of p70s6k (Weng et al., 1995). In addition, neutralizing antibodies raised against p70^^^ block PI3K mediated mitogenesis (Pullen and Thomas, 1997). PI3K therefore seems to lie upstream of p70^^*^, but PI3K does not directly phosphorylate p7 0s6k. Mutant forms of mTOR that do not bind the

rapamycin/FKBP12 complex overcome the inhibition of p70^^^ by rapamycin, suggesting that mTOR is also upstream of p70^^^ in vivo. It is not yet clear whether PI3K is upstream or downstream of mTOR, or whether the two act independently to activate p7Qs6k.

1.4.7.9.3 PKC and related kinases

Calcium independent isoforms of protein kinase C and protein kinase C related kinases have been found to be activated by 3’ phosphoinositides in vitro (Derman et al., 1997; Palmer et al., 1995; Toker et al., 1995). Although one study has shown that both PI(4,5)P2 and PI(3,4,5)P3 activate these kinases to the same extent, other studies report

conflicting data which might be attributed to differences in lipid presentation procedures. It was recently demonstrated that PI(3,4,5)Pg initiated cell motility of

NIH3T3 cells and that this effect was mediated by PKC (Derman et al.y 1997). Although the study was not able to distinguish which PKC family member or members were directly involved, data suggested that the PKCe was strongly regulated by these lipids. More recently it has been demonstrated that binding of the PH domain of PLCy to PtdIns(3,4,5)P3 in the membrane generated by PI3K in response to growth factor stimulation activates PLCyi (Falasca et al . y 1998).

1.4.7.9.4 Rab GTPases

The Rab proteins are members of the Ras superfamily of GTPases (Boguski and McCormick, 1993). There are more than 30 Rab isoforms present in mammalian cells, but they are specifically localised to vesicles at different stages of the exocytic or endocytic pathways (Simons and Zerial, 1993). There is some evidence that at least one Rab protein, Rab5, may be downstream of PI3K (Li et al . y 1995). In its GTP-

bound, active form, Rab5 stimulates fusion of early endosomes and thus increases the rate of endocytosis of various proteins (Simons and Zerial, 1993). Wortmannin inhibits this process and has also been shown to inhibit endosome fusion in vitro. In contrast, wortmanin does not inhibit endosome fusion downstream of constitutively active forms of Rab5, suggesting that PI3K miist lié upstream of Rab5. It has been suggested that PtdIns(3,4,5)P] is able, either directly or indirectly to stimulate nucleotide exchange on Rab5 (Li et al . y 1995).