Resultados del Estudio

protein kinase B isoforms.

The protein serine/threonine kinase protein kinase B (PKB, also known as Akt and R A C-a) is the cellular homologue of v-Akt, a protein encoded in the genome of the transforming retrovirus AKT-8 isolated from a rodent T-cell lymphoma (Bellacosa et a l, 1991). So far, three mammalian PKB isoforms (a , (3, and y) have been identified in mammals and one in D. melanogater. PKB proteins mediate the transduction of several important biological signals including cell survival (Dudek et al., 1997; Kauffmann Zeh et al., 1997) and glucose uptake/glycogen synthesis (Cross et al., 1995; Kohn et al., 1996; for review see Alessi and Cohen, 1998; Coffer et al, 1998).

All PKB proteins contain an N-terminal PH domain that when mutated blocks the PI 3- kinase-dependent activation of PKB by PDGF (Franke et al., 1997; Franke et al., 1995). PKB or the isolated PH domain binds Ptdlns(3,4)?2 with high affinity and causes dimérisation (Franke et al., 1997) and it has been reported that PtdIns(3,4)P2, but not PtdIns(4,5)P2 or PtdIns(3,4,5)P3, stimulates PKB activity in vitro (Franke et al., 1997; Freeh et al., 1997; Klippel et al., 1997). However, several other studies have not confirmed direct activation of PKB by these lipids (Alessi et al., 1997; James et al., 1996; Stokoe et al., 1997). The activation of PKBa by IGF-1 leads to the PI 3-kinase-dependent phosphorylation of two residues (T308 and S473, Alessi et al., 1996). Mutation of these residues to neutral amino acids abolishes P K B a activation and conversely, the introduction of acidic residues at these positions produces a constitutively activated molecule. PK B a phosphorylation at T308 is achieved by the 3-phosphoinositide- dependent kinase, PD K l. PDKl is directly activated by binding PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Alessi et al., 1997) and phosphorylation of PKB by PDKl is greatly enhanced in the presence of Ptdlns(3,4)?2 and Ptdlns(3,4,5)f3 micelles (Alessi et al.,

1997; Stokoe etal., 1997).

The studies outlined above have led to the following model for activation of PKB through PI 3-kinase: localised production of PtdIns(3,4)P2 at the plasma membrane recruits PKB and causes it to dimerise; this is thought to alter the conformation of the protein exposing it to phosphorylation by regulatory membrane-bound protein kinases including PDKl. PDKl phosphorylates a threonine residue in the activation loop of the PKB catalytic domain and once phosphorylated PKB becomes fully active leading to the phosphorylation of multiple substrates. Targets of PKB include glycogen synthase kinase GSK-3 (Cross et al., 1995), phosphofructo kinase PFK-2 (Deprez et al., 1997) which may be important in insulin signalling, BAD (Datta et al., 1997), and other unknown targets that lead to the inhibition of apoptosis (for review see Alessi and Cohen, 1998).

D-3 Pis are central to this model and PI 3-kinase has been implicated in generating the activating Ptdlns(3,4)7*2 and Ptdlns(3,4,5)f3 by using several experimental approaches. Firstly, mutant PDGF receptors which are unable to activate PI 3-kinase are also unable to

activate PKB (Burgering and Coffer, 1995). As mentioned above, low levels of wortmannin will block PKB activation by growth factors, as will dominant negative PI 3- kinase, whilst constitutively active PI 3-kinase leads to increased PKB activity, independent of growth factor stimulation (Klippel et al., 1996). Ptdlns(3,4)?2 is produced by the action of Ptdlns(3,4,5)?3 (5)-phosphatases in some cell types, however, there is also evidence for another pathway leading to the production of Ptdlns(3,4)?2- Recent studies in platelets suggest that the majority of Ptdlns(3,4)?2 generated following aggregation is synthesised by a route involving the phosphorylation of Ptdlns by the sequential actions of a Ptdlns 3-kinase and a PtdIns(3)P 4-kinase activity (Banfic et at., 1998a; Banfic et at., 1998b). This PtdIns(3)P 4-kinase activity could not be immuno- depleted with an anti-PtdlnsPK II mAh (Banfic et at., 1998a) and thus appears distinct from PtdlnsPK I la which can catalyse this reaction in vitro (Rameh et a i, 1997b; Zhang e ta l, 1997).

1 . 5 . 4 . b Guanine nucleotide e x c h a n g e factors a s

P td i n s (3 ,4 ,5 ) P 3 targets

Guanine nucleotide exchange factors (GEFs) catalyse the conversion of GTPases from the inactive GDP-bound to form the the active GTP-bound form. PH domains are found in all known GEFs which are specific for the Rho family of small GTPases (Section 1.10.1) and some GEFs specific for the Arf GTPases. In several cases D-3 phosphoinositides have been shown to interact with GEFs and subsequently stimulate their exchange factor activity.

The small GTPase Rac regulates the reorganisation of the actin cytoskeleton (Section 1.10.1) and is known to lie downstream of PI 3-kinase. Activation of PI 3-kinase by PDGF leads to an increase in levels of Rac-GTP (Hawkins et a l, 1995) although the precise mechanism is unknown. More recently it has been shown that the PH domain- containing Rac GEF, Vav can be stimulated by PtdIns(3,4,5)P3 (Han et a i, 1998) and thus Vav activation may be at least partially responsible for the effects of PI 3-kinase on GTP-Rac levels (Hawkins et a i, 1995).

Regulatory PH domains are also found in the N-terminus of ARNO and related nucleotide exchange factors Grpl and cytohesin (Chardin et a i, 1996; Klarlund et a i, 1997; Kolanus et a i, 1996). The Grpl and cytohesin PH domains have high affinities for Ptdlns(3,4,5)?3 and relatively low affinities for PtdIns(3,4,)P2 and PtdIns(3)P. ARNO also binds Ptdlns(3,4,5)?3 in vitro and its PH domain translocates to the plasma membrane in a wortmannin-dependent manner (Venkateswarlu et a i, 1998), suggesting that in vivo, Ptdlns(3,4,5)?3 is responsible for interaction with the PH domain. The finding that Ptdlns(4,5)7*2 binding to the PH domain of ARNO and stimulates the exchange of GDP for GTP on ARF-1 (Chardin et al., 1996) may be complicated by the

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