From the Inositide Laboratory, Department of Signaling, the Babraham Institute, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
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The GTPase Rac and the protein kinase B (PKB) are downstream targets of phosphatidylinositide 3OH-kinase in platelet-derived growth factor-stimulated signaling pathways. We have generated PAE cell lines inducibly expressing mutants of Rac. Use of these cell lines suggests that Rac is involved in both platelet-derived growth factor-stimulated membrane ruffling and the activation of p70S6K but not in the activation of PKB. Furthermore, expression of constitutively active alleles of PKB in PAE cells suggests that PKB is able to regulate the activity of p70S6K but not the cytoskeletal changes underlying membrane ruffling. Thus, our results indicate that Rac and PKB are on separate pathways downstream of phosphatidylinositide 3OH-kinase in these cells but that both of these pathways are involved in the regulation of p70S6K.
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INTRODUCTION |
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The PI3-kinase1 signaling pathway is widely used by receptors for growth factors, inflammatory stimuli, and antigens (1, 2). In the last few years, a number of PI3-kinase inhibition strategies have been used that have identified a family of downstream responses that are thought to be regulated by this pathway, including complex actions such as cell growth, differentiation, and movement (2). One of the current aims of research in this area is to understand the molecular mechanisms by which PI3-kinases engage the regulatory pathways associated with these responses.
The protein kinase PKB has previously been shown to be a ubiquitous target of PI3-kinase regulation in a variety of different contexts (3, 4). Very recently, good evidence has been provided that it is a direct molecular target for PtdIns(3,4,5)P3 and possibly PtdIns(3,4)P2, the phospholipids whose concentrations most immediately rise on agonist stimulation of PI3-kinases (5-8). This has suggested that PKB may be the primary signal transducer in the PI3-kinase signaling pathway, i.e. a role analogous to cAMP-activated protein kinase and the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel in their respective pathways. Evidence in favor of this idea comes from studies indicating that PKB can regulate other responses known to be controlled by PI3-kinase, such as glucose uptake/glycogen synthesis (9, 10), cell survival (for recent review, see Ref. 11), and p70S6K activation (4). Parallel work has suggested that the small GTPase Rac is also situated in a primary position in the PI3-kinase signaling pathway, since increased guanine nucleotide exchange on Rac is thought to drive PI3-kinase-dependent regulation of the cytoskeleton (known as lamellipodia formation/membrane ruffling) (12), superoxide formation (13), some secretory events (14), and also cell growth/transformation (15). A critical issue in understanding how the PI3-kinase signaling system operates is thus to understand whether PKB and Rac are activated in some form of hierarchy or as independent pathways. We have attempted to address this question in PDGF-stimulated PAE cells, where we have previously established that PI3-kinase-dependent membrane ruffling is dependent upon activation of Rac (16, 17).
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EXPERIMENTAL PROCEDURES |
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Antibodies-- Mouse mAb anti-HA (12CA5) was from Babco. Rabbit polyclonal anti-PKB serum was used as described (Burgering and Coffer (4)). Mouse mAb anti-Myc was a gift from Nick Ktistakis (Babraham Institute, UK). Mouse mAb anti-EE and rabbit polyclonal anti-Rac serum were obtained from ONYX Pharmaceuticals. Rabbit polyclonal anti-p70S6K antibody and the 32-mer p70S6K peptide substrate were gifts from Neil Anderson (University of Manchester, UK). Mouse mAb anti-Rac1 was from Transduction Laboratories (Lexington, KY).
Cell Culture-- Parental PAE cells expressing the PDGF receptor (Ref. 17; designated Wt-PAE cells in this study) were grown in F12 nutrient mixture (Ham F12; Life Technologies, Inc.) containing 10% heat-inactivated FBS (HI-FBS) at 37 °C in a 6% CO2 humidified atmosphere. Inducible Rac PAE cell lines were maintained in F12 nutrient mixture, 10% HI-FBS, 0.3 µg/ml puromycin and 60 µg/ml hygromycin.
Development of an Inducible Expression System-- We generated an inducible CMV promoter following the original development of IPTG-inducible SV40 promoters (18-20). This involved the insertion of lac repressor (LacI) binding sequences into the CMV promoter in a fashion whereby they did not significantly attenuate the efficacy of the promoter in the absence of LacI but were able to severely inhibit its action in its presence. Previous work with SV40 promoters indicated this would probably involve the insertion of multiple copies of LacI-binding sites near the TATA box. We used a convenient SacI site in the 2.1-kilobase pair CMV promoter to achieve this (Fig. 1A; there is another SacI site in the first intron of this promoter which we also exploited to insert further LacI-binding sites). We first constructed a simple transient expression vector, pCMV1-Luc, where expression of luciferase was under the control of the original CMV promoter ((21) a gift from A. Venkitaraman, LMB, Cambridge; cDNA encoding luciferase was obtained via pGEMLuc; Promega). We then ligated palindromic LacI-binding oligonucleotides (22) into the SacI sites of CMV1 under a range of different vector/oligo ratios. The products of the ligation were then used for transient expression in U937 cells together with an expression plasmid for LacI (pCMVlacI; see below). IPTG-inducible luciferase expression was monitored after 8 h in the presence or absence of 15 mM IPTG (Promega Luciferase assay kit). The sequence of the promoter (pCMV3) which gave the highest non-repressed and lowest repressed expression of luciferase is shown in Fig. 1A. We then added three further LacI-binding sites by ligating the SV40-based intron from pOPI3CAT (Stratagene) downstream of the CMV3 promoter (now denoted CMV3R; this followed advice from D. Wyborski, Stratagene, suggesting that, while not very effective in transient expression assays, this "R fragment" could produce good repression of the Rous sarcoma virus promoter in stable cell lines).
LacI Constructs-- The F9-1 promoter driving expression of LacI in p3'SS (Stratagene) was replaced with the CMV1 promoter to increase expression of nucleus-localized LacI ((23) pCMVLacI, available from Stratagene). pCMVLacI has a cassette conferring hygromycin resistance; for construction of the cell lines described here, this was replaced with a puromycin resistance cassette (derived from pBSpac (24) to make pCMVLacIpuro).
Generation of Stable PAE Cell Lines Inducibly Expressing Rac Mutants-- Clonal stable cell lines expressing nuclear-localized LacI were selected with puromycin (0.3 µg/ml) over a period of 8-10 days post-transfection with pCMVLacIpuro and isolated by ring cloning (while puromycin was continuously present in the medium). Clones expressing LacI protein were identified by immuno-cytostaining with a rabbit polyclonal anti-LacI serum (Stratagene) and expanded. One clone showing strong, nuclear-localized LacI expression (P23 PAE) was chosen for further use. All inducible lines described in this work were obtained from P23 PAE by a further round of transfection with pCMV3R(Rac) expression vectors (see below), selection (hygromycin, 60 µg/ml), and cloning. Screening for inducible expression of Rac mutants was done largely by Western blotting using the polyclonal rabbit anti-Rac serum, although in some experiments expression of the EE-tagged Rac mutants was quantified by anti-EE immunoprecipitations from [35S]methionine-labeled cells and SDS-PAGE (Fig. 1B) or by Western blot using a mouse mAb anti-Rac1 (Fig. 1C). Between 4 and 12 individual clones for each Rac mutant were finally selected for expansion and storage. For a number of the experiments presented here, more than one Rac mutant was investigated of each type, with essentially identical results.
Rac Constructs-- The cDNAs encoding N-terminally EE-tagged Wt-Rac, N17Rac (12), and V12Rac (12) were obtained from ONYX Pharmaceuticals. Standard cloning procedures were used to ligate these cDNAs under the CMV3R promoter (see above) in a vector containing an expression cassette conferring hygromycin resistance (pA71d; a gift from A. Venkitaraman, LMB, Cambridge).
PKB Constructs--
The cDNA encoding bovine PKB (from
Boudewijn M. T. Burgering and Paul J. Coffer, Utrecht, The
Netherlands) was ligated into a pCMV transient expression vector with
either N-terminal EE (MEEEEFMPMEF) or Myc (MEQKLISEEDLEF) tags
(generated by standard polymerase chain reaction-based strategies). The
single point mutants (R25C, W99L, T308A, T308D, S473A, S473D, and
K197A) and the
PH(1-125) mutants of PKB were generated by a
polymerase chain reaction-based mutagenic strategy using Pfu
polymerase (Stratagene) and low cycle numbers (
16). PLC
PH-PKB was
generated using Stratagene's Seamless Cloning Kit®;
residues 6-99 of PKB were replaced with residues 7-121 of PLC
(Roger Williams, LMB, Cambridge), creating a PH domain swap at the
conserved C-terminal tryptophan. The resulting construct was ligated
into pCMV transient expression vector with either an N-terminal EE or
Myc tag. Myrist.-PKB containing the Yes myristoylation consensus at the
N terminus (MGLCIKSKEDKSM) (25) and Gag-PKB (4) were ligated into pCMV
transient expression vector with C-terminal EE or Myc tags. All
constructs were sequenced to verify that they contained no polymerase
chain reaction-generated errors. In some experiments, HA-tagged Wt-PKB
(4) was transiently expressed using an SV40 promoter-based expression
vector (pSG5, Stratagene).
Transient Transfection-- Wt-PAE cells or N17Rac-PAE cells were transfected with pCMV-PKB mutants by electroporation at 250 V and 960 microfarads (1 × 107 cells in 500 µl) essentially as described for U937 cells (26). Transfected cells were grown on glass coverslips (for immunofluorescence experiments) or in 6-well tissue culture plates (for kinase assay) in F12, 10% HI-FBS for 7-30 h and then serum-starved for 15 h in F12, 1% HI-FBS or F12, 0.1% fatty acid-free BSA (see the figure legends).
PKB Assay--
For PKB assays (4), tissue culture dishes were
washed twice in 25 mM Hepes-NaOH, pH 7.3, 1.8 mM CaCl2, 5.37 mM KCl, 0.81 mM MgSO4, 112.5 mM NaCl, 25 mM glucose, 1 mM NaHCO3, 0.1%
fatty acid-free BSA and left to prewarm in a 37 °C waterbath for 5 min before being stimulated with the indicated concentrations of PDGF for 5 min. In some cases, cells were treated with 100 nM
wortmannin (stock in Me2SO) for 10 min at 37 °C prior to
activation. After cell activation, the medium was aspirated followed by
immediate addition of 1 ml of ice-cold PKB lysis buffer (50 mM Hepes, 5 mM -glycerophosphate, 120 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 15 mM sodium
pyrophosphate, pH 7.5) and incubation on a gently rocking ice bath for
5 min. Cells were then scraped into Eppendorf tubes and centrifuged at
8,000 × g for 5 min. The supernatant was precleared
with protein G-Sepharose beads (pre-equilibrated in PKB lysis buffer
containing 1% BSA) for 30 min and then incubated for 2 h with mAb
anti-EE or mAb anti-HA as appropriate (non-covalently pre-coupled to
protein G-Sepharose or protein A-Sepharose, respectively). The beads
were washed 3 times in PKB lysis buffer and twice in 50 mM
Hepes, 10 mM MgCl2, pH 7.4. The reaction was
carried out in a final volume of 50 µl in 50 mM Hepes, pH
7.4, 10 mM MgCl2, 50 µM MgATP, 2 µM cyclic AMP-dependent protein kinase
inhibitor (Sigma), 10 µg of myelin basic protein, and 5 µCi of
[
-32P]ATP at 30 °C for 20 min. It was stopped by
the addition of 50 µl of 2× Laemmli sample buffer and boiling for 5 min. Proteins were separated by 15% SDS-PAGE, and
-32P-labeled myelin basic protein was visualized and
quantified with a Bio-Rad phosphorimager. In experiments where the
activities of several different EE-tagged PKB mutants were measured,
their expression levels were compared in parallel by Western blotting. For this, the transfected cells were grown and serum-starved under the
same conditions as those for the PKB assay. Then, equal numbers of
cells from each transfection were solubilized in Laemmli SDS sample
buffer, proteins separated by 8% SDS-PAGE, tranferred to nitrocellulose, and blotted with mAb anti-EE. Expression of the PKB
mutants was visualized by the standard ECL procedure. Autofluorograms were densitometrically scanned with a Bio-Rad Gel Doc 1000 system and
quantified with the Bio-Rad Molecular Analyst program to estimate the
differences in expression levels. The PKB activity of each mutant was
then corrected for its average expression level in all presented
experiments.
p70S6K Assay--
For p70S6K assays
(27), the cells were activated, and solubilization was done as
described for PKB assays except that the p70S6K lysis
buffer was made of 55 mM Tris, pH 8.0, 132 mM
NaCl, 22 mM NaF, 1.1 mM EDTA, 5.5 mM EGTA, 11 mM sodium pyrophosphate, 1%
Nonidet P-40. The solubilized material was precleared with protein
A-Sepharose beads (pre-equilibrated in p70S6K lysis buffer
containing 1% BSA) for 30 min, incubated for 2 h on ice with
rabbit polyclonal anti-p70S6K antibody, and then with
pre-equilibrated protein A-Sepharose beads for 1 h. The beads were
washed twice in ice-cold p70S6K lysis buffer and twice in
"p70S6K buffer" (25 mM MOPS, pH 7.2, 1 mM EDTA, 0.05% Triton X-100, 10 mM
dithiothreitol, 20 mM para-nitrophenol
phosphate). The samples were prewarmed to 30 °C for 3 min in 50 µl
of p70S6K buffer containing 2.5 µM cyclic
AMP-dependent protein kinase inhibitor and 3.5 µg of
32-mer p70S6K substrate peptide. The reaction was started
by addition of 10 µl "ATP solution" per sample (80 mM
MgCl2, 200 µM MgATP, and 3 µCi of
[-32P]ATP). Incubation was carried out at 30 °C for
15 min and stopped by the addition of orthophosphoric acid to a final
concentration of 200 mM. Samples were spotted onto Whatman
P81 paper, washed 6 × 5 min in 175 mM orthophosphoric
acid, and quantified by scintillation
-counting.
Immunofluorescence Microscopy-- Cells for immunofluorescence microscopy were grown on glass coverslips and serum-starved as described above. They were then pretreated or not with 100 nM wortmannin for 10 min and stimulated with the indicated concentrations of PDGF for 5 min at 37 °C. Activation was stopped by aspirating the medium and immediate addition of 4% paraformaldehyde in 100 mM Pipes, pH 7.2, 2 mM EGTA, 2 mM MgCl2 for 15 min at room temperature. The cells were washed three times in 150 mM Tris, pH 7.2, permeabilized for 10 min at room temperature in PBS, 0.1% Triton X-100, and then washed twice in PBS. In some experiments, filamentous actin was directly labeled with TRITC-phalloidin (Sigma; 1:100-1:400 dilution of 200 µg/ml stock in methanol) in PBS, O.5% BSA for 1 h at 37 °C. In other experiments, the coverslips were first blocked with PBS, O.5% BSA for 20 min at room temperature and then incubated with an appropriate dilution of mAb-anti-Myc (1:100) or anti-PKB antibody (1:400) for 1 h at 37 °C. The coverslips were washed 4 × for 5 min in PBS, O.5% BSA and then incubated with an appropriate dilution (usually 1:100) of secondary antibody fluorescein isothiocyanate goat anti-mouse or fluorescein isothiocyanate-goat anti-rabbit (both Sigma) and TRITC-phalloidin (as above) concomitantly. Coverslips were washed 3 × for 5 min in PBS, O.5% BSA, 5 min in PBS, and then in H2O before being mounted onto microscope slides with Aqua-polymount anti-fading solution (PolySciences Inc., Northampton, UK).
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RESULTS |
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Lamellipodia Formation and Membrane Ruffling in PAE Cells Stably
Expressing Rac Mutants--
To investigate the role of Rac in the
PI3-kinase signaling pathway, we have generated stable PAE cell lines
that inducibly express Wt-Rac (CL-25), constitutively active V12Rac
(CL-84), or dominant negative N17Rac (CL-23), using a strategy that is detailed under "Experimental Procedures" (Fig.
1). These cell lines expressed low basal
levels of the exogenous Rac mutants (10% of endogenous Rac) but, in
all cases, the expression increased dramatically upon addition of
increasing concentrations of IPTG (Fig. 1, B and
C). The sensitivity of induction to IPTG varied between
individual clones, but the maximal level of expression of heterologous
Rac was between approximately 5-fold (CL-23 N17Rac line) and
approximately 20-fold (CL-25 Wt-Rac line) above endogenous Rac levels
(Fig. 1C).
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Rac Activates p70S6K but Not PKB-- Earlier reports have shown that both PKB and p70S6K are downstream targets of PI3-kinase (3, 4, 29), and studies using transient transfection of fibroblasts with N17Rac and p70S6K have indicated that Rac mediates the activation of p70S6K by PDGF (30). We used the inducible Rac-PAE cell lines to study the effects of Wt-Rac, V12Rac, and N17Rac expression on the PDGF-stimulated activation of PKB and p70S6K in PAE cells (Fig. 3). In parental PAE cells, PDGF stimulated PKB and p70S6K activation was about 10- and 2.5-fold over basal, respectively, and the activation of both enzymes was wortmannin-sensitive.
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PKB Is Activated via Its PH Domain and by Phosphorylation-- Recent work has suggested that PKB may be a direct target of PtdIns(3,4,5)P3 via the binding of the lipid to its PH domain (3, 31). The activation of PKB by PtdIns(3,4,5)P3 is complex, however, and also involves PI3-kinase-dependent phosphorylation of at least two sites, Thr-308 and Ser-473 (32). A Thr-308-directed PKB kinase has been recently characterized (6-8) that may be directly regulated by PtdIns(3,4,5)P3 (8) and is probably at least partially responsible for PDGF-stimulated phosphorylation of this site in vivo. To situate PKB in the PDGF signaling pathway in PAE cells, we looked next at the PDGF-stimulated activation of a range of phosphorylation site and PH domain mutants of PKB transiently transfected into Wt-PAE cells (Fig. 4, A and B).
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Constitutively Active Mutants of PKB--
The data shown in Fig. 3
have suggested that Rac is not involved in the activation of PKB in
PDGF-stimulated PAE cells, and those shown in Fig. 4, A and
B, are consistent with the idea that PKB is an immediate
target of PI3-kinase in this pathway. This leaves two possibilities,
PKB could either be situated upstream of Rac or parallel to Rac. To
study whether PKB controls Rac, we constructed three PKB cDNAs that
might encode constitutively active PKB alleles, PH-PKB (described in
Fig. 4B), Gag-PKB (the oncogenic viral version (34)) and
myrist.-PKB which carries the membrane targeting
myristoylation/palmitoylation consensus of Yes fused to its N terminus
(35). Gag-PKB and myrist.-PKB have been shown to act as active PKBs in
recent studies, supposedly because both are myristoylated and
consequently membrane-targeted (33, 34, 36).
PKB Does Not Induce Lamellipodia Formation and Membrane Ruffling-- We used the constitutively active PKB mutants to study their effect on the Rac-mediated response of lamellipodia formation and membrane ruffling. We transiently transfected Wt-PAE cells with Wt-PKB, the constitutively active PKB mutants, or kinase-dead PKB, stimulated them with PDGF, and analyzed the effects on lamellipodia formation and membrane ruffling by immunofluorescence microscopy after double staining for the exogenous PKB proteins and filamentous actin (Fig. 5).
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PKB Activates p70S6K Independently of Rac-- We have shown above that both Rac and PKB can activate p70S6K (Figs. 3A and 4D). To investigate further the relationship between these three enzymes within the PDGF pathway, we used the N17Rac line CL-23 to see whether PKB-mediated activation of p70S6K is dependent or independent of Rac activity. For this, CL-23 cells were transiently transfected with kinase-dead PKB or the constitutively active Gag-PKB. Gag-PKB induced a 2-3-fold activation of p70S6K (Fig. 6). Induction of dominant negative N17Rac with IPTG did not make a significant difference to this response, showing that PKB-mediated activation of p70S6K is independent of Rac activity.
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DISCUSSION |
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We studied here the relationship between Rac and PKB in the PDGF-stimulated PI3-kinase signaling pathway in PAE cells. Inducible expression of Rac mutants had no effect on PDGF-stimulated activation of PKB, suggesting that PKB is positioned upstream of or parallel to Rac in this pathway. Active PKB mutants were not sufficient to cause Rac-dependent lamellipodia formation and membrane ruffling, suggesting that Rac and PKB are activated in parallel.
The generation of cell lines that stably and inducibly express constitutively active or dominant negative forms of Rac has allowed us to show that Rac activity is both necessary and sufficient for lamellipodia formation/membrane ruffling and for the activation of p70S6K. As Rac is situated downstream of PI3-kinase in the PDGF signaling pathway, wortmannin would not be predicted to inhibit the activation of p70S6K by expression of V12Rac. Interestingly, Chou and Blenis (30) and we (data not shown) have found, however, that wortmannin does partially inhibit the ability of V12Rac to activate p70S6K. We do not understand the mechanism of this effect, but the use of wortmannin as a tool to inhibit PI3-kinases is widespread and generally accepted to be quite specific, with relatively few alternative targets in the nanomolar range. This suggests that PI3-kinases or related proteins that are wortmannin-sensitive are involved in the activation of p70S6K by Rac (one possible mechanism is the involvement of an autocrine loop in these cells, dependent upon Rac).
We have used a range of PKB mutants to show that an intact PH domain
and the capacity to phosphorylate Thr-308 and Ser-473 are required for
PDGF-stimulated activation of PKB in PAE cells, confirming reports in
other systems (3, 32, 33). We then created a range of constitutively
active PKBs (i.e. the PH mutant and the membrane-targeted
mutants Gag-PKB and myrist.-PKB) and used these to show that PKB
activity is sufficient for the activation of p70S6K but not
for lamellipodia formation and membrane ruffling. A mechanism of action
for these constitutively active PKB mutants has been suggested by
others (33), namely membrane targeting followed by increased
phosphorylation and consequent activation. In agreement with this, we
have found that Thr-308 mutants of myrist.-PKB are inactive (data not
shown). We have also found that Thr-308 mutants of
PH-PKB are
inactive (data not shown). It is surprising that the deletion of the PH
domain should lead to increased phosphorylation of Thr-308 and
consequent PKB activation. However, these observations are consistent
with a model whereby the phosphorylation of PKB is regulated by PH
domain-mediated translocation to the membrane and also a PH
domain-controlled inhibition of Thr-308 phosphorylation (6).
As mentioned above, the use of the constitutively active mutants has demonstrated that PKB activity is not sufficient for lamellipodia formation and membrane ruffling, i.e. further targets of PI3-kinase are involved in this response, beyond those dedicated to the activation of PKB. We cannot, however, rule out a contributory role for PKB in this pathway. This would require an effective PKB inhibition strategy. The literature contains reports of the use of kinase-dead PKB as a dominant negative allele to block the effect of endogenous PKB on cell survival pathways (38); unfortunately, our use of this construct results in the reduction in expression of reporter constructs (e.g. heterologously expressed PKB or p70S6K) or has no measurable change in the PDGF-stimulated activity of the relevant endogenous proteins (we have selected populations of cells transiently expressing kinase-dead PKB by co-transfection with green fluorescent protein expression vectors and fluorescence-activated cell sorting; data not shown).
When we transfected PKB mutants into PAE cells, we found that, in the
starved and resting cells, Wt-PKB, PH-PKB, and kinase-dead PKB were
mostly localized in the nucleus and only partially in the cytoplasm,
whereas earlier reports have indicated that although the oncogenic
viral form or Gag-PKB is nuclear, non-transforming versions are
cytoplasmic (36, 39). Recent work has also shown that, in 293 cells,
overexpressed HA-tagged Wt-PKB is cytosolic in resting cells and
translocates into the nucleus upon cell stimulation with insulin-like
growth factor-1 (40). We cannot, at present, explain the differences
between our results and these studies. The nuclear localization that we
observed was not simply due to pCMV-driven overexpression, since we
obtained the same localization when expressing lower amounts of
heterologous PKB (using pS5G-driven HA-tagged PKB; cells stained with
mAb anti-HA), and when we labeled endogenous PKB with rabbit polyclonal
anti-PKB serum (data not shown). Furthermore, the predominantly nuclear
localization of heterologously expressed PKB was not restricted to PAE
cells, as we observed it equally in COS, HepG2, or Chinese hamster
ovary cells transfected with Myc-tagged PKB with various techniques (DEAE-dextran, liposomes, or calcium phosphate; data not shown). These
observations suggest that PKB might have a role as a nuclear signaling
enzyme, in addition to its putative role at the plasma membrane.
Our data have shown that PKB and Rac are activated separately in a PI3-kinase-controlled pathway. Although PKB seems to be a direct target of PtdIns(3,4,5)P3, it is very likely that further direct targets are involved in the activation of Rac. Other analogous second messenger-generating signaling systems (e.g. cAMP or inositol 1,4,5-trisphosphate) operate through a very small number of primary targets for the messenger molecule. It is possible, however, that PtdIns(3,4,5)P3 may act via its interaction with membrane-targeting PH domains (41) to regulate a larger number of proteins in parallel, reflecting an increased emphasis on positional information as a major regulatory function of the messenger molecule.
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ACKNOWLEDGEMENT |
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We acknowledge Boudewijn Burgering for helpful
discussions and the generous gifts of rabbit polyclonal anti-PKB serum
and cDNA encoding bovine PKB.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received postdoctoral fellowships from the European Community
Commission.
§ Fellow of the Biotechnology and Biological Sciences Research Council. To whom correspondence should be addressed. Tel.: 44-1223-832 312; Fax: 44-1223-836 481; E-mail: Phillip.Hawkins{at}bbsrc.ac.uk.
1
The abbreviations used are: PI3-kinase,
phosphatidylinositide 3OH-kinase; BSA, bovine serum albumin; HI-FBS,
heat-inactivated fetal bovine serum; LacI, lac repressor;
PDGF, platelet-derived growth factor BB; PKB, protein kinase B, Akt;
PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)trisphosphate;
PtdIns(3,4)P2, phosphatidylinositol (3,4)diphosphate;
p70S6K, p70 ribosomal S6 kinase; mAb, monoclonal antibody;
HA, hemagglutinin; CMV, cytomegalovirus; IPTG,
isopropyl-1-thio--D-galactopyranoside; PAGE,
polyacrylamide gel electrophoresis; Pipes,
1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline;
TRITC, tetramethyl rhodamine isothiocyanate; MOPS,
4-morpholinepropanesulfonic acid.
2 B. Burgering, personal communication.
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REFERENCES |
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