The PI3K-Akt signaling module

1.3.1. Phosphatidylinositol 3-kinase (PI3K)

There are multiple isoforms of PI3Ks in mammalian cells, and the best characterized is class I. Class I PI3Ks comprise a p110 catalytic subunit and a regulatory adapter subunit: p85.

The p85 subunit contains an SH3 domain and two SH2 domains (127; 128). These SH2 domains allow the interaction between p85 and proteins containing specific tyrosine-phosphorylated sequences, such as the ones present on tyrosine-phosphorylated FAK proteins. This process recruits the p110 catalytic subunit to the plasma membrane, where it can phosphorylate phosphatidyl inositol-4,5-biphosphate [PI(4,5)P₂] and thereby generate phosphatidyl inositol-3,4,5-triphosphate [PI(3,4,5)P3] (129). This phosphorylated lipid is implicated in signaling pathways involved in the regulation of cell survival, gene expression and cell metabolism, as well as cytoskeletal rearrangements (130; 131). It has been shown that the PI3K-generated [PI(3,4,5)P₃] lipids recruit signaling proteins by direct binding to their PH domains, and either allosterically modify their activity or induce relocalization of the protein to defined areas of the plasma membrane, where activation can occur (132). Such signaling proteins include the serine-threonine kinase Akt (133; 134). Not all but a major part of PI3K signaling is mediated via Akt. The alternative signaling pathways mediated by PI3K are out of the scope of this introduction and are reviewed by Downward (135).

1.3.2. Protein kinase B (PKB/Akt)

In 1991, several groups independently identified the serine/threonine kinase Akt (also called PKB) (136-138). One of these groups identified this kinase as being the product of the oncogene v-akt of the acutely transforming retrovirus AKT8 found in a rodent T-cell lymphoma (136). Three isoforms of Akt exist in mammalian cells: Akt1, Akt2 and Akt3. These three Akt proteins, although encoded by distinct genes localized on different chromosomes, have approximately 80% amino acid identity and similar domain structures (139). Akt1 and Akt2 both show a wide distribution of expression, however their levels of expression differ (140; 141). Akt family proteins contain a central kinase domain with specificity for serine or threonine residues

PI3K-generated [PI(3,4,5)P₃] lipids may recruit Akt to the plasma membrane, where it is phosphorylated and activated by a membrane-associated protein has been proposed by Alessi and colleagues (145; 146). This model has been reinforced by the isolation of this putative [PI(3,4,5)P₃]-dependent protein kinase (PDK-1) that phosphorylates and activates Akt on residue Thr-308 (147). The putative kinase(s) or mechanism(s) responsible for Ser-473 phosphorylation have not yet been clearly identified (148). Nevertheless, several candidates have been proposed to be Ser-473 kinases, including integrin-linked kinase (ILK) (149).

In summary, Akt is activated (indirectly) by the PI3K-generated [PI(3,4,5)P3] lipids, and its activity is thus sensitive to the specific PI3K inhibitors Wortmannin and LY294002 (150). Full activation of Akt requires phosphorylation at the residues Thr-308 and Ser-473.

1.3.3. Activation of the PI3K-Akt pathway by ECM

It is now known that a diverse array of physiological stimuli, including ECM, are capable of inducing Akt kinase activity, primarily in a PI3K-dependent manner (139). Diverse ECM molecules (including fibronectin, laminin and collagen) and integrin subunits have been shown to induce PI3K and/or Akt activities. As mentioned above, the p85 subunit of PI3K can associate directly with FAK in response to cell adhesion to the ECM protein fibronectin (151).

Furthermore, it was shown in the same report that PI3K activity associated with FAK increases upon attachment to fibronectin and that this is concomitant with increased FAK tyrosine phosphorylation (151), providing a first indication that PI3K might be involved in the signaling downstream of cell adhesion-induced FAK activation. The same group subsequently reported that phosphorylation of FAK on residue Tyr-397 is required for its binding to PI3K and for the activation of PI3K (108). Furthermore, adhesion of cells to fibronectin leads to accumulation of PI3K-generated phosphorylated lipids, resulting in activation of Akt kinase activity (152). This fibronectin-induced activation of Akt is dependent on PI3K activity (152). Generation of primary fibroblasts carrying a targeted deletion of the segment of β1 integrin cytoplasmic domain required for activation of FAK has been reported (74). These fibroblasts have impaired FAK-PI3K-Akt signaling in response to adhesion to fibronectin (75). Therefore, it can be concluded that engagement of integrins to cognate ECM can activate the PI3K-Akt signaling pathway.

1.3.4. Role of the PI3K-Akt pathway in cell survival

Yao and Cooper provided the first evidence indicating that PI3K is implicated in the suppression of apoptosis (153). This report demonstrated that inhibition of PI3K activity using two pharmacological inhibitors of PI3K (LY294002 and Wortmannin) abrogates the ability of NGF to promote cell survival. Since, PI3K activity has been found to be required for growth factor-dependent survival of a wide variety of cultured cell types ranging from fibroblasts to neurons (139). The observation that Akt is a target of PI3K, together with the finding that PI3K

mediates growth factor-dependent survival, suggested that Akt might be a critical regulator of cell survival. This hypothesis has been confirmed by many groups (as reviewed by (139)), leading to the conclusion that the PI3K-Akt pathway is one of the major anti-apoptotic pathways operating in cells (139). In addition, it has been shown that specific deletion of Akt1 leads to partial neonatal mortality and the surviving Akt1 knock-out animals are smaller in size than control mice, suggesting that Akt1 may be involved in regulation of survival and/or proliferation of cells (154; 155). Several effectors downstream of activated Akt have been reported to mediate its survival signaling. For example, Akt has been shown to directly phosphorylate the pro-apoptotic Bcl2-family member Bad, leading to its binding to the 14-3-3 protein and thus preventing it from exerting its pro-apoptotic effects (156-158). Furthermore, Akt has been reported to regulate the activity of transcription factors such as members of the Forkhead family of transcription factors and NF-κB (159; 160), leading to altered gene expression. However, it seems that these effects of Akt on the activity and/or expression of proteins regulating apoptosis are cell-type and/or context-dependent.

The PI3K-Akt pathway was first reported to mediate ECM-induced survival of normal epithelial cells in a study showing that overexpression of constitutively active PI3K and Akt mutants are able to protect MDCK cells against anoikis (161). Since, several groups have confirmed that the PI3K-Akt pathway can mediate the pro-survival signaling from ECM and/or integrins (69; 72; 162). The induction of the PI3K-Akt pro-survival signaling by ECM and/or integrins has been reported to occur specifically on a given ECM substrate or through a specific integrin subunit, and this specificity is cell type-dependent (72; 73). For example, it has been shown that overexpression of the α5 integrin subunit protects epithelial cells against serum deprivation-induced apoptosis through the PI3K-Akt pathway, whereas overexpression of α2 subunit does not (72). The pro-survival signaling emanating from ECM-integrin interaction or FAK activation does not always involve PI3K-Akt pathway (122; 123). Indeed, other pathways such as JNK (123) and MAPK-ERK (73) have also been reported to mediate the pro-survival signaling from ECM.

In summary, it has been shown that ECM and/or engagement of integrins to cognate ECM proteins can activate the PI3K-Akt survival signaling pathway. However, as discussed

1.4. The MAP kinase ERK pathway