Perspective

Akt Signaling--Linking Membrane Events to Life and Death Decisions

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Science  31 Jan 1997:
Vol. 275, Issue 5300, pp. 628-630
DOI: 10.1126/science.275.5300.628

Modules made of protein kinases control cellular processes. This discovery—perhaps the most important in signal transduction research during the past 5 years—is typified by growth factor stimulation of the Ras-Raf-MAP kinase module (1). One of the many initial events that occur after growth factors bind to their cognate growth factor receptor tyrosine kinases (RTKs) is the recruitment and activation of the phosphoinositide 3-kinases (PI 3-kinases). Inositol lipids ph osphorylated at the D3 position by PI 3-kinases act as second messengers somewhat analogous to cyclic adenosine 3',5'-monophosphate (cAMP) and calcium. The serine/threonine protein kinase Akt (also called protein kinase B or PKB), identi fied first as an oncogene, is one of the major targets of PI 3-kinase-generated signals (25). Results on pages 661 and 665 of this issue of Science and elsewhere (610) now provide new information on the mechanism of signal propagation from RTKs to Akt and reveal that Akt may participate in gro wth factor maintenance of cell survival.

Crucial to the discovery of Akt (1113) and its function was the recognition that Akt is a proto-oncogene (12) and the characterization of its pleckstrin homology (PH) domain (14). The recognition that PH domains can bind lipids suggested a mechanism linking the activation of PI 3-kinase and Akt activity (6, 9, 10, 15). PI 3-kinase activity i s potently inhibited by wortmannin and by the structurally unrelated inhibitor LY294002. Both of these inhibitors can block the rapid activation of Akt (up to 50-fold) by platelet-derived growth factor, epidermal growth factor, basic fibroblast growth fac tor, insulin, and insulin-like growth factor-1 (IGF-1) (25, 16). Activation of Akt by protein phosphatase inhibitors (16) is, however, relatively insensitive to wortmannin and LY294002, indicating that it is the lipid kinase activity of PI 3-kinase that mediates Akt activation by growth factors. These and other data place Akt firmly downstream of PI 3-kinase.

How does the generation of the PI 3-kinase-derived second messengers, phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and PtdIns-3,4,5-P3, promote activation of Akt? The current data indicate three steps: (i) translocation of t he kinase to the membrane, (ii) attachment to the membrane by means of PH domain binding to phospholipid, and (iii) phosphorylation (see figure). The importance of translocation is emphasized by recent experiments (17, 18) indicating that targeting of Akt to the membrane by the addition of a myristoylation motif to the NH2-terminus can promote activation of the kinase by a mechanism that is resistant to wortmannin or LY294002 inhibition. Constructs lack ing a PH domain but with the membrane-attachment myristoylation motif are activated to a similar extent as the native protein. This result not only indicates that the PH domain functions primarily to anchor the protein at the membrane, but it also suggest s that the oncogenic potential of v-akt arises from creation of a myristoylation site at the NH2-terminus and the consequent constitutive kinase activity. The high-affinity association of Akt with PtdIns-3,4-P2 and PtdIns-3, 4,5-P3 not only provides a means for attaching the kinase to the membrane but also, as shown by Franke et al. (6) and elsewhere (9, 10), in the case of PtdIns-3,4-P< >2, promotes a conformational change leading to an increase of kinase activity.

Growth factor-promoted activation of Akt requires PI 3-kinase.

Growth factor binding promotes recruitment and activation of PI 3-kinase after autophosphorylation of the receptor on tyrosine residues. PI 3-kinase at the membrane converts P tdIns-4,5-P2 (PI-4,5-P2) to PtdIns-3,4,5-P3 (PI-3,4,5-P3), and PtdIns-3,4-P2 (PI-3,4-P2) is generated by inositol 5'-polyphosphatase. Akt is recruited to the membrane, undergoes a conformational change, and become s phosphorylated (activated). Subsequently, Akt is released from the membrane to phosphorylate specific targets. Activation by IGF-1 also required insulin receptor substrate 1 (IRS1) (shown in white). KD, kinase domain; RD, regulatory domain.

However, the in vitro activation of Akt by PtdIns-3,4-P2 (two- to sixfold) is modest compared with that induced by growth factor stimulation. The two phospholipids PtdIns-3,4-P2 and PtdIns-3,4-P3, which bind at micromolar concentrations to the PH domain, are normally not detectable in unstimulated cells (19) but accumulate transiently upon cell stimulation by growth factors. It is likely that this transient accumulation promotes the association of Akt with the membrane. What is the source of this PtdIns-3,4-P2? The fact that wortmannin inhibits activation suggests that PtdIns-3,4-P2 is derived from PtdIns-3,4,5-P3 (the product of PI 3-kinase) by a phospholipid phosphata se (see figure). Both the recently described growth factor-stimulated inositol polyphosphate 5'-phosphatases (20) and the type II PI 3-kinases (21) could be important components in the overall regulation of PtdIns-3,4-P2 amounts in the membrane.

PI 3-kinase activity ultimately leads to an increase in Akt kinase by promoting its phosphorylation, in addition to the direct allosteric effect of the lipid products of PI 3-kinase (9, 16). Insulin and IGF-1 promo te a 20- to 50-fold activation of kinase activity and phosphorylation of Akt at two sites: Thr308 in the T-loop and Ser473 on the COOH-terminal regulatory domain. Both phosphorylation events can be inhibited by wortmannin in vivo. An alysis of a mutant, kinase-dead Akt, revealed that Thr308 and Ser473 are phosphorylated by an upstream kinase (PKBK). The amino acid sequences around these two residues suggest that two distinct PKBKs are involved. Furthermore, the r esults of membrane targeting experiments indicate that the PKBKs are constitutively localized at the membrane. The scene is now set for the isolation of this upstream kinase, likely an effort of several laboratories.

It was proposed recently that inositol trisphosphate (IP3), presumably generated from PtdIns-4,5-P2 by phospholipase C-γ, could release PH domain-containing proteins from membranes (15); release of Akt from the membrane by IP3 could also be a key regulatory step. After its release, Akt would become available to phosphorylate downstream targets until inactiv ated by dephosphorylation by protein phosphatase 2A (PP2A). The search for authentic in vivo substrates of Akt is of intense interest, given the number of physiological processes regulated by PI 3-kinase. The first identified physiological substrate of Ak t was glycogen synthase kinase 3 (GSK3). Phosphorylation of GSK3 leads to inactivation of this kinase and stimulation of glycogen synthesis (5). The regulation of GSK3 by Akt is also likely to affect other aspects of cellular function because of its role in the regulation of protein synthesis, modulation of transcription factors (AP-1 and cAMP response element-binding protein) and the tumor suppressor adenomatous polyposis coli (APC), cell fate determination (in Drosophila), a nd dorsoventral patterning (in Xenopus) (5, 22). Other data suggest that Akt is upstream of the p70 ribosomal protein s6 kinase but the connection is likely to be indirect (3).

The new data by Dudek et al. (7) demonstrate that Akt is important for the survival of cerebellar neurons. Thus the “orphan” kinase has now moved to center stage as a crucial regulator of life and death decisions ema nating from the cell membrane. Previous data (23) indicated that survival of several cell lines is PI 3-kinase-dependent, and the new results show that IGF-1 protects cerebellar neurons from apoptosis (cell death) by activating Akt. T his conclusion is based on the authors demonstration that expression of exogenous Akt enhances cell survival by dramatically reducing apoptosis and that transfection of dominant negative mutant kinase constructs (kinase-dead or PH domain) inhibited surviv al of neurons promoted by IGF-1.

Nerve growth factor (NGF) also promotes Akt activation in pheochromocytoma PC12 cells, suggesting that kinase activation is also involved in the survival promoted by NGF (24). The implication from the new work is that the Akt signalin g pathway is able to prevent apoptosis of neurons after NGF treatment. Does activated Akt protect cells from programmed cell death promoted by other stimuli and physiological conditions? Interestingly some reports (25) indicate growth factor-induced neurite outgrowth in PC12 cells is inhibited by wortmannin, suggesting yet another role for Akt.

Are other PH domain-containing protein kinases (about 12 at the last count) also recruited to the membrane and activated by PI 3-kinase? Only time will tell if they are all as interesting as Akt.

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