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Dual Role of Phosphatidylinositol-3,4,5-trisphosphate in the Activation of Protein Kinase B

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Science  25 Jul 1997:
Vol. 277, Issue 5325, pp. 567-570
DOI: 10.1126/science.277.5325.567

Abstract

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

PKB (also known as c-Akt or Rac-PK) was originally identified as the transforming oncogene in a retrovirus from a spontaneous thymoma in an AKR mouse (1). The oncogene product contains all of the coding sequence of PKB, with an NH2-terminal fusion 60 bases upstream of the initiator methionine to the viral gag gene (2). This fusion creates a myristoylation signal, which causes a relocation of PKB from the cytosol to the plasma membrane (3). This altered localization is likely to explain the deregulated oncogenic activity of the viral fusion protein because targeting PKB to the plasma membrane confers constitutive activity (4). PKB contains a pleckstrin homology (PH) domain at its NH2-terminus, a domain that has been implicated in binding inositol lipids (5). PKB appears to be activated as a consequence of increased phosphoinositide 3-kinase (PI3K) activity in cells stimulated with mitogens, because chemical inhibitors of PI3K or dominant-negative subunits of PI3K both block the activation of PKB (6-8), and transfection of cells with a constitutively active form of PI3K causes activation of PKB (9). Potential targets of PKB include glycogen synthase kinase–3 (10) and p70 ribosomal protein S6 kinase (6). PKB is also crucial in mediating cell survival (11). The exact mechanism of activation of PKB is not completely understood. Activation is usually accompanied by phosphorylation (12), and incubation of activated PKB with serine-threonine phosphatases abolishes activity (4). However, the lipid products of PI3K may also bind (13) and activate (7, 14, 15) PKB. Furthermore, an intact PH domain is necessary for activation of PKB by growth factors and constitutively active PI3K (7, 9), but this has been disputed (4). We therefore examined the relative contributions of lipid binding and phosphorylation of PKB on its activation in a defined in vitro system.

Lysates from COS1 cells transiently transfected with hemagglutinin (HA)-tagged PKB (16) were incubated with synthetic dipalmitoyl PtdIns(3,4,5)P3[(dipalmitoyl)-sn-phosphatidyl-d-myo-inositol-(3,4,5)-trisphosphate]. Addition of either PtdIns(3,4,5)P3 or an adenosine triphosphate (ATP)–regenerating system to the cell lysate had little effect on the activity of PKB that was subsequently immunoprecipitated and assayed (Fig. 1). Similarly, if PKB was first immunoprecipitated through the HA tag and PtdIns(3,4,5)P3 was added either alone or in combination with ATP to the immunoprecipitate, there was no activation of PKB. However, if both PtdIns(3,4,5)P3 and the ATP-regenerating system were added to the cell lysate, PKB that was subsequently affinity-purified had increased kinase activity, both toward itself and an exogenous substrate, histone h2B (Fig. 1A). For optimal activation of PKB, lipid, ATP, and detergent were all required. Greater activation was achieved in vitro after addition of PtdIns(3,4,5)P3 and ATP than after stimulation of cells with epidermal growth factor, but in both cases the activity was abolished by incubation of the affinity-purified PKB with a serine-threonine–specific protein phosphatase [Fig. 1A and (17)]. Three different sources of the dipalmitoyl form of the biological isomer of PtdIns(3,4,5)P3 all had a similar effect on PKB. However, a stearoyl-arachidonoyl–substituted version of PtdIns(3,4,5)P3[(1-stearoyl,2-arachidonoyl)-sn-phosphatidyl-d-myo-inositol-(3,4,5)-trisphosphate] was the most potent in the activation of PKB (17), and because this is likely to be the major species of PtdIns(3,4,5)P3 within the cell (18), this lipid was used for further characterization of the events involved in the activation of PKB under these conditions (19).

Figure 1

Identification of an activity that phosphorylates and activates PKB only in the presence of PtdIns(3,4,5)P3. (A) Lysate from COS1 cells transiently expressing HA-PKB was incubated with 50 μM PtdIns(3,4,5)P3 (PIP3), an ATP-regenerating system [5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, creatine kinase (50 μg/ml)], 1% NP-40, or combinations of these as indicated (lanes 1 through 5). The immunoprecipitated PKB was then assayed for kinase activity with histone h2B as a substrate. In lanes 7 and 8 the PKB was first immunoprecipitated, and the washed immunoprecipitate (ip) was incubated with PtdIns(3,4,5)P3 and an ATP-regenerating system and then assayed with histone h2B as a substrate. The sample in lane 6 is from an identical experiment to that shown in lane 5, except that after immunoprecipitation, PKB was incubated with 200 U of lambda phosphatase (New England Biolabs) for 30 min. The phosphorylated PKB and histone h2B bands are indicated by arrows. Molecular size markers are in kilodaltons. (B andC) Brain cytosol (20 mg) was fractionated by chromatography on a Mono Q column (Pharmacia). Each fraction was assayed for the ability to cause phosphorylation (B) or activation (C) of purified recombinant Glu-Glu–tagged (26) PKB in the presence (circles) or absence (squares) of PtdIns(3,4,5)P3.

The above results indicated that both PtdIns(3,4,5)P3binding to either PKB or an upstream component, together with phosphorylation of PKB, are required for full activation of PKB. To dissect these possibilities, we chromatographed lysates from a number of different rat tissues on a MonoQ column and tested fractions for their ability to phosphorylate and activate purified PKB in a PtdIns(3,4,5)P3- and ATP-dependent manner, which would reflect the presence of an activator or upstream kinase (20). Activity from rat brain cytosol was resolved into two peaks of activity that phosphorylated PKB. The peak eluting at 475 mM NaCl phosphorylated PKB both in the absence and presence of PtdIns(3,4,5)P3 and was present in all rat tissues tested. However, the peak eluting at 150 mM NaCl phosphorylated PKB exclusively in the presence of PtdIns(3,4,5)P3. This latter activity overlapped with, but did not exactly coincide with, an activity that activated PKB, also in a PtdIns(3,4,5)P3-dependent manner (Fig. 1C) (21). The amount of the PtdIns(3,4,5)P3-sensitive PKB kinase activity varied in cytosol from eight different tissues tested, with brain and thymus being the richest sources (17).

Phosphoamino acid analysis of the phosphorylated PKB revealed that threonine was the only amino acid phosphorylated under these conditions (Fig.2A). Phosphopeptide mapping showed that the majority of the [32P]phosphate was incorporated into a single phosphopeptide under these conditions (Fig. 2B), which also contained only phosphothreonine (17). Two residues of PKB are phosphorylated in cells stimulated with growth factors, Thr308 and Ser473 (12). We therefore mutated either of these residues to alanine or aspartate and examined the effect of these mutants in allowing phosphorylation and activation by the active fractions from Fig. 1. Mutant PKB in which Ser473 was changed to Ala (S473A) was activated to the same extent as wild-type PKB, a mutant in which Ser473 was changed to Asp (S473D) was activated to a greater extent, whereas PKB with mutations at Thr308 was not activated by the upstream kinase (Fig. 2C). Mutations at Ser473 also had little effect on the phosphorylation of PKB mixed with active fractions, whereas mutations at Thr308 inhibited phosphorylation of PKB (Fig. 2D). The residual phosphorylation of the Thr308 mutants resulted from the increased phosphorylation of the two minor phosphopeptides in wild-type PKB (17). Mutation of the ATP-binding site of PKB (Lys179 to Ala) eliminated its ability to phosphorylate an exogenous substrate (Fig. 2C), but it was still phosphorylated when mixed with active fractions in a PtdIns(3,4,5)P3-dependent manner to the same degree as wild-type PKB (Fig. 2D). Tryptic and V8 proteolytic digestion of the phosphorylated PKB, followed by Edman degradation of the separated phosphopeptides in a spinning cup sequenator (22), also gave results indicative of Thr308 being the major site of phosphorylation (Fig. 2E).

Figure 2

Threonine-308 is the phosphorylation site responsible for the activation of PKB by PtdIns(3,4,5)P3 and the upstream kinase. (A) Phosphorylated PKB (0.5 μg, 4500 cpm) was subjected to partial acid hydrolysis, and the products were resolved by two-dimensional thin-layer chromatography. The phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards are indicated. (B) Phosphorylated PKB (4 μg, 60,000 cpm) was digested with trypsin, and the peptides were resolved on a Supelco C18 column with an increasing gradient of acetonitrile. Fractions (0.8 ml) were collected and counted for 32P. A single major peak eluting at 35% acetonitrile and two minor peaks eluting at 16 and 21% acetonitrile were obtained. (C andD) Purified recombinant PKB (0.5 μg) containing the indicated mutations was assayed for activation (C) or phosphorylation (D) by Mono Q fractions 5 and 6 in the presence or absence of PtdIns(3,4,5)P3 as indicated (A, Ala; D, Asp; K, Lys; S, Ser; T, Thr). (E) Phosphorylated PKB (4 μg, 60,000 cpm) was digested with either trypsin or V8, and the peptides resolved on the C18column. The major peak from each digest was subjected to automated Edman degradation in a spinning cup sequenator and each cycle counted for 32P. The sequence surrounding Thr308 is shown, and the predicted cleavage sites are indicated by arrows. The second peak of radioactivity at cycle 7 in the tryptic peptide is probably due to incomplete digestion.

The above results suggested that the role of PtdIns(3,4,5)P3 could either be to promote activation of the kinase that phosphorylates PKB on Thr308, or to bind to PKB itself and change its conformation to allow phosphorylation of Thr308 by the upstream kinase. Because phospholipids bind specifically to the PH domain of PKB (13, 15), we assessed the effects of PH domain mutations and truncations on phosphorylation and activation in this system. Two mutations in the PKB PH domain, Trp99 to Leu and Arg25 to Cys, abolished the phosphorylation and activation of PKB by the upstream kinase in the presence of PtdIns- (3,4,5)P3 (Fig. 3, A and B). This finding suggested the possibility that the PH domain of PKB could be masking the ability of the upstream kinase to phosphorylate Thr308, and that binding of PtdIns(3,4,5)P3 might relieve this constraint. A PKB mutant lacking the first 125 amino acids (ΔPH-PKB) was phosphorylated and activated by the upstream kinase in the absence of PtdIns(3,4,5)P3 (Fig. 3D). However, the ability of the upstream kinase to phosphorylate and activate ΔPH-PKB was still enhanced in the presence of PtdIns(3,4,5)P3(Fig. 3D). This suggests that PtdIns (3,4,5)P3 has two functions in the activation of PKB—direct binding to the PH domain of PKB, which allows phosphorylation by the upstream kinase, and direct activation of the upstream kinase itself.

Figure 3

Requirement of the PH domain of PKB for phosphorylation and activation by the upstream kinase in a PtdIns(3,4,5)P3-dependent manner. (A and B) HA-tagged PKB containing the indicated mutations was transiently expressed in COS1 cells and affinity-purified by using the HA tag (WT, wild type; C, Cys; L, Leu; R, Arg, W, Trp). The immunoprecipitate was added to Mono Q fractions 5 and 6 and assayed for phosphorylation (A) or activation (B) by these fractions. Equivalent amounts of PKB were assessed as judged by protein immunoblotting [(A) lower panel]. Molecular size markers in (A) are in kilodaltons. (C andD) Brain cytosol (20 mg) was fractionated by Mono Q, and each fraction was assayed for the ability to phosphorylate and activate purified recombinant (C) wild-type PKB or (D) a PKB mutant lacking the first 125 amino acids (ΔPH-PKB), in the presence or absence of PtdIns(3,4,5)P3.

Further evidence for this dual mode of regulation was provided by examining the lipid specificity for phosphorylation and activation of full-length PKB and the ΔPH-PKB mutant by the upstream kinase. The biologically relevant stereoisomer of PtdIns(3,4,5)P3 (termed DD-PIP3) had a median effective concentration (EC50) of about 3 to 5 μM in stimulating phosphorylation and activation of full-length PKB, whereas its enantiomer [(2-arachidonoyl, 3-stearoyl) sn-phosphatidyl-l-myoinositol-(3,4,5)-trisphosphate, termed LL-PIP3] was essentially inactive (Fig.4A). However, the EC50 of DD-PIP3 in stimulating phosphorylation and activation of ΔPH-PKB was 0.3 to 0.5 μM, and its enantiomer also activated to a smaller extent (Fig. 4B). PtdIns(3,4)P2 caused activation and phosphorylation (albeit only at high concentrations) of full-length PKB, but it was inactive at causing phosphorylation or activation of the ΔPH-PKB mutant (Fig.4C). Activation and phosphorylation by PtdIns (3,4)P2 was dependent on the presence of the upstream kinase (17). PtdIns(4,5)P2 or a diacetyl form of PtdIns(3,4,5)P3 were both unable to cause phosphorylation or activation of either full-length or truncated PKB (17).

Figure 4

Similar but distinct lipid specificities of the PKB PH domain and the upstream kinase. (A) Wild-type PKB (0.5 μg) or (B) the ΔPH-PKB mutant (0.5 μg) was incubated with 10 μl of Mono Q fractions 5 and 6 and various amounts of either the biological form of PtdIns(3,4,5)P3 (DD-PIP3) or its enantiomer (LL-PIP3) as indicated. Activation (upper panels) and phosphorylation (lower panels) of PKB was assessed. (C and D) Wild-type PKB (0.5 μg) or the ΔPH-PKB mutant (0.5 μg) was incubated with various concentrations of dipalmitoyl PtdIns(3,4)P2, and activation (C) and phosphorylation (D) of PKB was determined.

Deletion of the PH domain of PKB has been reported to either enhance the activity of PKB (4, 23) or to have no effect on its basal or stimulated activity (4), depending on the cell type tested. The discrepancy in these results may reflect the amount or distribution of the upstream kinase in various cell types. The upstream kinase was more sensitive to PtdIns(3,4,5)P3 than PKB, and PtdIns(3,4)P2 did not activate this kinase (Fig. 4); therefore the effect of PtdIns(3,4)P2 is likely to be on PKB itself, an effect previously noted by other groups (14,15).

These results suggest that the PtdIns(3,4,5)P3 signal can be transduced by the actions of a PtdIns(3,4,5)P3-activated protein kinase; thus, the regulation of this pathway is analogous to that of other signaling systems that respond to small molecule signals (adenosine 3′,5′-monophosphate–dependent protein kinase, protein kinase C, and Ca2+/calmodulin-dependent protein kinases). In the PtdIns(3,4,5)P3 pathway, the lipid signal controls PKB activity in two distinct but cooperative ways, which is reminiscent of the role of adenosine monophosphate (AMP) to allosterically activate the AMP-activated protein kinase, and also to activate the kinase that phosphorylates the AMP-activated protein (24). This may ensure tight regulation of PKB at the correct membrane localization, and it will be interesting to see whether this dual regulatory principle will apply to other targets in the PI3K signaling pathway.

Note added in proof: A PtdIns(3,4,5)P3-dependent protein kinase has been purified (25) that may be related to the upstream kinase described here.

  • * Present address: University of California, San Francisco Cancer Research Institute, 2340 Sutter Street, San Francisco, CA 94115, USA.

  • To whom correspondence should be addressed. E-mail: stokoe{at}cc.ucsf.edu

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