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© The Rockefeller University Press,
0021-9525/1999//1511 $5.00
The Journal of Cell Biology, Volume 145, Number 7,
, 1999 1511-1520
Regular Articles |
The Dynamics of Protein Kinase B Regulation during B Cell Antigen Receptor Engagement
Signal Transduction Laboratories, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom
This study has used biochemistry and real time confocal imaging of green fluorescent protein (GFP)-tagged molecules in live cells to explore the dynamics of protein kinase B (PKB) regulation during B lymphocyte activation. The data show that triggering of the B cell antigen receptor (BCR) induces a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of activated B cells. Hence, PKB has three potential sites of action in B lymphocytes; transiently after BCR triggering PKB can phosphorylate plasma membrane localized targets, whereas during the sustained B cell response to antigen, PKB acts in the nucleus and the cytosol. Membrane translocation of PKB and subsequent PKB activation are dependent on BCR activation of phosphatidylinositol 3-kinase (PI3K). Moreover, PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes. However, under conditions of continuous PI3K activation or BCR triggering there is only transient recruitment of PKB to the plasma membrane, indicating that there must be a molecular mechanism to dissociate PKB from sites of PI3K activity in B cells. The inhibitory Fc receptor, the Fc
RIIB, mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase SHIP into the BCR complex. Herein we show that coligation of the BCR with the inhibitory FcRIIB prevents membrane targeting of PKB. The FcRIIB can thus antagonize BCR signals for PKB localization and prevent BCR stimulation of PKB activity which demonstrates the mechanism for the inhibitory action of the FcRIIB on the BCR/PKB response.
Key Words: Akt/PKB • phosphatidylinositol 3-kinase • GSK3 • B cell antigen receptor • FcRIIb
Abbreviations used in this paper: BCR, B cell antigen receptor; GFP, green fluorescent protein; GSK3, glycogen synthase kinase 3; PH, plextrin homology; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B.
Address correspondence to Doreen Cantrell, Imperial Cancer Research Fund, Lymphocyte Activation Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Tel.: 44-171-269-3307. Fax: 44-171-269-3479. E-mail: d.cantrell{at}icrf.icnet.uk
FOREIGN antigen binding to the B cell antigen receptor (BCR)1 triggers the activation of cytosolic tyrosine kinases including Syk and lyn and BTK (Yamanashi et al., 1992; Desiderio and Siliciano, 1994; Rawlings et al., 1996; DeFranco, 1997). These tyrosine kinases then initiate a cascade of signaling pathways that control B cell function during an immune response. The BCR regulates the metabolism of inositol phospholipids by two pathways: BCR stimulation of phospholipase C results in the hydrolysis of phosphatidylinositol (4,5) biphosphate [PI(4,5)P2] and the production of inositol 1,4,5-triphosphate which initiates an increase in intracellular calcium (Cambier and Jensen, 1994). PI(4,5)P2 breakdown simultaneously produces diacylglycerol which activates serine/threonine kinases of the protein kinase C family. BCR triggering also stimulates the activity of phosphatidylinositol 3-kinase (PI3K) which phosphorylates PI(4,5)P2 on the D-3 position of the inositol ring to produce PI(3,4,5)P3 (Gold et al., 1992). D-3 phosphoinositides bind to the plextrin homology (PH) domains of proteins and either allosterically modify their activity or induce relocalization of the protein to defined areas of the plasma membrane where activation can occur (Rameh et al., 1997). Genetic evidence for the importance of PI3K for B lymphocytes has been illustrated by the phenotype of mice lacking expression of the p85
regulatory adapter protein for PI3K which show profound defects in B cell function (Fruman et al., 1999; Suzuki et al., 1999).
During B cell activation both positive and negative regulatory signaling cascades are vital for a balanced immune response and immune homeostasis. One important negative feedback mechanism that operates in B cells is mediated by the FcRIIb and the src homology 2 (SH2) domain containing inositol 5' phosphatase (SHIP) (Takai et al., 1996; Ono et al., 1997; Tridandapani et al., 1997; Helgason et al., 1998; Okada et al., 1998; Sarao et al., 1998). Coligation of the BCR with the FcRIIB by antigen–antibody complexes results in tyrosine phosphorylation of an immune cell tyrosine based inhibitory motif (ITIM) in the FcRIIB (Burshtyn and Long, 1997). The SH2 domain of SHIP can bind to the phosphorylated ITIM, thereby recruiting this inositol 5' phosphatase into the BCR/FcRIIB complex. SHIP dephosphorylates PI (3,4,5)P3 to produce PI(3,4)P2 and accordingly, coligation of the BCR with FcRIIB diminishes BCR elevation of intracellular PI(3,4,5)P3. PI3K controlled signaling pathways are thus the proposed primary targets for FcRIIb inhibition (Hippen et al., 1997; Scharenberg et al., 1998; Scharenberg and Kinet, 1998).
Recent studies have identified two proteins regulated by PI3K and SHIP in B cells: a tyrosine kinase Btk (Bolland et al., 1998; Scharenberg et al., 1998) and a serine/threonine kinase protein kinase B or Akt/PKB (Aman et al., 1998; Jacob et al., 1999), the cellular homologue of the transforming oncogene of the murine retrovirus Akt8 (Staal and Hartley, 1988). PKB has been shown to play an important role in maintenance of cell survival in fibroblasts and epithelial cells (Zha et al., 1996; Kauffmann-Zeh et al., 1997; Kulik et al., 1997). In T cells, PKB can stimulate the activity of E2F transcription factors which are important components of the mechanisms that control the mammalian cell cycle (Brennan et al., 1997). PKB also phosphorylates and inactivates GSK3 (Cross et al., 1995), an enzyme initially identified as a regulator of glycogen metabolism but which also has broader functions: interactions between GSK3 and β-catenin (Rubinfeld et al., 1996) regulate activity of the T cell factor–lymphoid enhancer factor (Tcf-Lef). GSK3 can also control the nuclear export of nuclear factor of activated T cells (NFAT), transcription factors involved in cytokine gene induction (Beals et al., 1997).
In this report we have explored the dynamics of PKB regulation by the BCR and the inhibitory FcRIIB in B lymphocytes. We used green fluorescent protein (GFP; Heim and Tsien, 1996; Rizzuto et al., 1996) to tag PKB and time lapse confocal microscopy of live cells to reveal that BCR regulation of Akt/PKB has the following sequence: a rapid and transient recruitment of Akt/PKB to the plasma membrane followed by a sustained activation of the enzyme. Analysis of the location of GFP-tagged PKB indicated that active PKB is not normally maintained at the plasma membrane but is found in the cytosol and nucleus. We show that PI3K signals are both sufficient for membrane recruitment of Akt/PKB and for sustained activation of the enzyme. BCR activation of PKB is subject to a negative feedback control mechanism initiated by the FcRIIB and mediated by SHIP (Aman et al., 1998). We show that coligation of the BCR with the inhibitory FcRIIB prevents membrane localization of PKB. These data illustrate the importance of PI3K-mediated signals in controlling the cellular localization of PKB and they reveal the mechanism for the inhibitory action of the FcRIIB on the BCR/PKB response.
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and the pan anti-PKB were from Upstate Biotechnology, the pan anti-GSK3 antisera were from Santa Cruz Biotechnology. Rabbit antisera reactive with the COOH-terminal residues 904–918 of PKD/PKCµ were generated by standard protocols. Anti–sheep and anti–goat HRP-conjugated antibodies were obtained from Chemicon, and anti–mouse HRP and anti–rabbit HRP were from Sigma Chemical Co. The rat CD2 monoclonal antibody OX34 and the 12CA5 monoclonal reactive with the Ha epitope tag were purified from hybridoma supernatants by standard protocols. The FcRIIB blocking antibody (FcBlockTM) was purchased from PharMingen. The PI3K inhibitor LY294002 was purchased from BIOMOL Research Laboratories, the histone H2B was from Boehringer Mannheim, the protein kinase inhibitor and ATP were from Sigma Chemical Co., and the [-32P]ATP was from Amersham.
A chimeric protein of the extracellular and transmembrane domain of the rat CD2 molecule fused with the p110a catalytic subunit of PI3K (rCD2p110) targets PI3K to the membrane and creates a constitutively active enzyme that generates PI(3,4,5)P3 and PI(3,4)P2. The rCD2p110 construct and a catalytically inactive form with a mutated ATP-binding site (rCD2-p110R/P) were described previously (Reif et al., 1996, 1997). The GFP-tagged PH domain of PKB and its non–lipid-binding R25C mutated form were generated, respectively, from subcloning HindIII/BamH1 from pCMV6 containing HA-tagged murine PKB PH domain (HA-AH) or HA-R25C (Franke et al., 1995) into -C1 (Clontech), the BsrG1/Sal1 fragment was then replaced by a linker of six glycine residues. Full length PKB was COOH-terminally GFP-tagged using standard protocols. Oligonucleotides introduced in pEGFP (Clontech) a sequence corresponding to the COOH-terminal sequence of PKB from the BclI site to the end of the protein at the NH2 terminus of GFP and a BglII site and a stop codon at the COOH terminus of GFP. This resultant fragment was purified and ligated to pSG5 HA-PKB digested with BclI and BglII. All constructs were verified by DNA sequencing. The GFP-PKB fusion protein undergoes activation in response to BCR ligation (data not shown).
Cell Stimulation and Transfection
A20 cells were washed and resuspended at 2 x 107/ml in RPMI 1640. The cells were incubated at 37°C with either 10 µg/ml of anti–mouse F(ab')2 fragment or 15 µg/ml of intact Ig, for the indicated periods of time. Some samples were preincubated with 5 µM of Ly294002 or 2.5 µg/ml anti-FcRIIb for 30 min before stimulation. Cells were quickly pelleted at 4°C and lysed in 50 mM Hepes, 10 mM NaF, 10 mM iodoacetamide, 75 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM Na2VO3, and 1 µg/ml of each for leupeptin, pepstatin A, and chymotrypsin. Nuclear debris were eliminated by 20 min centrifugation at 14,000 rpm and the supernatant proteins precipitated for 1 h at –70°C in 1.5 vol acetone. Precipitates were pelleted by 20 min of centrifugation at 14,000 rpm, resuspended in Laemmli buffer, boiled, and fractionated on 10% SDS-PAGE, with the exception of samples for GSK3 blotting, which were analyzed on 7.5% gels. Immunoblotting was performed by standard protocols and revealed by chemiluminescence (ECL; Amersham). For transfection, 500 µl of cells was aliquoted with DNA, electroporated at 310 V and 960 µF using a Bio-Rad gene pulser. Thereafter cells were cultured for 12–14 h before stimulation, microscopic analysis, or lysis.
Immunoprecipitation and Kinase Assay
5 x 106 cells transfected with HA-tagged PKB were stimulated as indicated and lysed in 120 mM NaCl, 50 mM Hepes, 10 mM NaF, 1 mM EDTA, 40 mM β-glycerophosphate, 1% NP-40, 1 mM Na2VO3, and 1 µg/ ml of each for leupeptin, pepstatin A, and chymotrypsin. After elimination of nuclear debris, the supernatant was cleared by addition of 1/25 vol of a 30% solution of Sepharose-coupled protein G. Supernatants were incubated for 2 h with 10 µg/ml of the anti-HA antibody 12CA5, and another 45 min after addition of 1/10 vol of the Sepharose–protein G solution. Pellets were washed once in lysis buffer, twice in 500 mM LiCl, 100 mM Tris, pH 7.5, 1 mM EDTA, and once in assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, and 1 mM DTT). Dried samples were assayed by incubation for 30 min at room temperature with 2.5 µg H2B, 5 µM protein kinase inhibitor, 50 µM ATP, and 3 µCi of [-32P]ATP in a final volume of 15 µl. Boiled samples in Laemmli buffer were fractionated by SDS-PAGE. The level of H2B phosphorylation was analyzed by exposing the lower part of the gel on a sensitive film, and the amount of PKB protein immunoprecipitated was determined by Western blot analysis with a pan PKB antibody.
Nuclear Fractionation
After stimulation, cells were disrupted in 10 mM Hepes, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 1 mM Na2VO3, 0.15% NP-40, and the nuclear fraction pelleted at 14,000 rpm for 1 min. Cytosolic extracts were removed and NaCl was added to obtain a final concentration of 120 mM. These cytosolic extracts were then concentrated by acetone precipitation. The nuclear pellets were washed three times with lysis buffer and then resuspended in 20 mM Hepes, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.42 M NaCl, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 1 mM Na2VO3, 0.15% NP-40, and lysed for 30 min at 4°C. Debris was eliminated by 20 min of centrifugation at 14,000 rpm and supernatants, corresponding to nuclear extracts, were acetone precipitated.
Confocal Time Lapse Imaging
After transfection, cells were resuspended at 106/ml in 35-mm glass bottom dishes (MatTek Corp.). Before analysis dishes were gently rinsed with warm Hanks' medium without phenol red. Dishes were mounted in the warm chamber of the confocal microscope (Zeiss Laser Scanning Microscope 5.10). Samples were excited at 488 nm by an argon laser and detected with a 63 x 1.4 NA oil immersion objective. The first image was recorded just before stimulus addition directly into the dish, and then scans were made automatically every 5 s by using Zeiss LSM software. Images shown are representative of a minimum of five experiments. In Fig. 2, anti-FcRIIB and LY294002 were added to the dishes in Hanks' medium 30 min before microscopic analysis.
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RIIB inhibits BCR signal transduction pathways by recruiting the inositol 5' phosphatase SHIP into the BCR complex (Ono et al., 1997; Tridandapani et al., 1997). The data in Fig. 1 A show that B cell activation with intact IgG, which cross-links the BCR with the FcRIIB, fails to stimulate PKB phosphorylation or PKB catalytic activity as judged by in vitro (Fig. 1 B) and in vivo (Fig. 1 C) assays of PKB function. In the presence of an anti-FcRIIB antibody which blocks the interaction of IgG with the FcRIIB receptor, intact IgG is able to trigger the BCR without coligating inhibitory FcRIIB into the BCR complex. The presence of the blocking antibody to the FcRIIB then allows intact IgG to ligate the BCR and stimulate PKB activity (Fig. 1).
PKB Localization in B Lymphocytes
Membrane localization of PKB is known to be essential for the activation of the enzyme but there are conflicting reports as to whether activated PKB is present in the nucleus or maintained at the cell membrane (Andjelkovic et al., 1997; Meier et al., 1997; Watton and Downward, 1999). To examine the localization of PKB carefully in intact B cells we expressed a GFP-tagged construct of wild-type PKB in A20 cells and analyzed the cellular localization of this enzyme in quiescent and BCR-activated cells. Membrane targeting of PKB is mediated by interactions of the PH domain with either PI(3,4,5)P3 or PI(3,4)P2. Accordingly, we examined also the cellular localization of a GFP-tagged PKB-PH domain. Fig. 2 shows midsection confocal images of A20 cells transiently transfected with GFP wild-type PKB or the GFP-tagged PKB-PH domain. In unstimulated A20 cells, GFP-PKB was uniformly distributed throughout the cytosol of the cell and was also present in the nucleus. The GFP-tagged PKB-PH domain of PKB was similarly distributed. Western blot analysis of nuclear extracts prepared from A20 cells revealed that endogenous PKB was present in the nuclei of these lymphocytes (Fig. 3).
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A striking feature of the effect of the BCR on PKB membrane localization is its speed and transience. Kinetic analysis of the effect of the BCR on PKB activity reveals that this is also a rapid response detectable within 30 s of BCR triggering (data not shown). The data in Fig. 2 B show that the effect of BCR ligation is to induce a stable activation of PKB in a response that is maximal at 1 min and sustained for at least 60 min (Fig. 2 B). It is also clear that BCR induced phosphorylation of GSK3; an endogenous substrate for PKB is a response that is maintained for a period of 1–2 h (Fig. 2 B). In summary, analysis of PKB cellular localization and activity shows that quiescent B cells express PKB in the cytosol and the nucleus. BCR triggering induces rapid membrane localization and activation of PKB. PKB targeting to the membrane in response to BCR ligation is very transient and can be detected within 10 s but is finished by 40–50 s. In contrast, the stimulatory effects of the BCR on PKB activity are sustained for at least 1 h. 1 min after BCR triggering there is no discernible plasma membrane localized PKB, rather the active PKB is present in either the cytosol or the nucleus.
Active PKB Is Found in the Nucleus and Cytosol of BCR-triggered Cells
The question of the localization of active PKB is important because it affords insight as to the potential site of action of this kinase. To determine whether active PKB is present in the nuclear or cytosolic compartment of B cells, A20 cells were triggered via the BCR and cytosolic and nuclear cell fractions were resolved by SDS-PAGE and analyzed by Western blot with the phospho-PKB antibody that recognizes active enzyme or with the pan PKB antisera. These data (Fig. 3) show that endogenous PKB is found in both the nucleus and cytosol of quiescent or activated B cells. In cells activated by cross-linking the BCR with F(ab')2 fragment of anti–mouse IgG there was a strong reactivity of both cytosolic and nuclear PKB with the phospho-PKB antisera. These results show that active PKB is found in both the cytosol and nucleus of activated B lymphocytes.
PI3 Kinase Signals Are Essential and Sufficient for Membrane Localization and Activation of PKB in B Cells
PI3K signals are essential and sufficient for activation of PKB in T lymphocytes (Reif et al., 1997). A comparison of the data in Fig. 4, A and B, shows that the BCR-induced transient membrane localization of GFP-PKB is abolished by the PI3K inhibitor Ly294002. The data in Fig. 4 C show that Ly294002 also prevents BCR activation of PKB. Hence, the ability of the BCR to regulate the cell localization and activity of PKB is dependent on PI3 kinase activity. To determine whether PI3K signals are sufficient for membrane targeting and activation of PKB we examined the effects of expression of constitutively active PI3 kinase on PKB cellular localization and activity. The BCR stimulates the activity of a PI3K complex that comprises a regulatory p85 and a catalytic p110 subunit. A chimeric protein of the extracellular and transmembrane domain of the rat CD2 molecule fused with the p110 catalytic subunit of PI3K (rCD2p110) targets PI3K to the membrane and creates a constitutively active enzyme that generates PI(3,4,5)P3 and PI(3,4)P2 when expressed in cells. The data in Fig. 5 show that expression of the constitutively active PI3K, rCD2p110, induces a strong activation of PKB. In these experiments A20 cells were transfected with increasing concentrations of the rCD2p110 expression vector resulting in increasing levels of expression of rCD2p110. The effect of the active rCD2p110 constructs on endogenous PKB activity was monitored using the phospho-PKB antisera. The results (Fig. 5 A) show that there was a dose-dependent stimulation of PKB activity by the expressed active PI3K. This stimulatory effect of rCD2p110 was dependent on the kinase activity of the chimera since expression of a kinase inactive mutant, rCD2p110 R/P, did not stimulate PKB activity. Moreover, treatment of A20 cells with the PI3K inhibitor LY294002 abrogated PKB activity in cells expressing rCD2p110 (Fig. 5 B).
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Coligation of the BCR with the FcRIIb Prevents Membrane Localization of PKB
BCR activation of PKB is subject to a negative feedback control mechanism initiated by the FcRIIB and mediated by SHIP (Fig. 1) (Aman et al., 1998). The stimulation of PKB catalytic function requires membrane localization of the kinase (Franke et al., 1997; Frech et al., 1997; Klippel et al., 1997) plus its phosphorylation at two residues: threonine 308 and serine 473 by PI3K-dependent protein kinases (Alessi et al., 1997; Alessi and Cohen, 1998; Bellacosa et al., 1998; Stephens et al., 1998). The FcRIIB/SHIP complex will influence both the kinetics and magnitude of PIP3 levels during B cell activation and could remove a membrane targeting signal for PKB. However, SHIP dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2 which is also able to bind to the PH domain of PKB (Franke et al., 1997; Frech et al., 1997). Thus, it is possible that PKB will still be recruited to the plasma membrane when the BCR is coligated with the FcRIIB complex and that SHIP terminates PKB-mediated responses by preventing the phosphorylation and activation of membrane-localized PKB. For example, osmotic stress can prevent PKB activation without preventing membrane localization of Akt/PKB (Meier et al., 1998). To study the effects of BCR coligation with the FcRIIB on the cellular localization of PKB, A20 cells expressing the GFP-PKB constructs were stimulated with either F(ab')2 fragment of anti–mouse IgG or with intact IgG. Confocal images of a midsection of the cells were then taken at 5-s intervals after addition of the stimulus. The results in Fig. 7 show that B cell activation with intact IgG, which cross-links the BCR with the FcRIIB, fails to stimulate PKB membrane translocation. Fig. 7 shows confocal images of cells 15 s after BCR triggering but cells were monitored for several minutes and no membrane localization of PKB could be seen in BCR/FcRIIB coligated cells at any time point. In the presence of an anti-FcRIIB antibody which blocks the interaction of IgG with the FcRIIB receptor, intact IgG is able to trigger the BCR without coligating inhibitory FcRIIB into the BCR complex. The results show that presence of the blocking antibody to the FcRIIB then allows intact IgG to induce plasma membrane localization of PKB.
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The FcRIIb mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase, SHIP, into the BCR complex. The importance of the FcRIIB/ SHIP complex for B cell homeostasis is illustrated by the phenotype of mice lacking expression of either FcRIIB or SHIP which are prone to inflammatory disease and show a higher sensitivity to anaphylaxis (Takai et al., 1996; Helgason et al., 1998). FcRIIB ligation with the BCR prevents activation of PKB; this response was revealed by quantitation of PKB activity in vitro but was confirmed also by analysis of the effects of BCR and FcRIIB coligation on the phosphorylation of GSK3, an endogenous substrate for PKB. SHIP dephosphorylates PI(3,4,5)P3 to produce PI(3,4)P2 and is essential for the inhibitory action of the FcRIIB (Ono et al., 1997). The PH domain of PKB, which mediates membrane targeting of the enzyme, is able to bind the product of SHIP, PI(3,4)P2, in vitro. Nevertheless, the present data show that coligation of the BCR with the inhibitory FcRIIB prevents membrane targeting of PKB. This was an intriguing result that reveals a mechanism for the inhibitory action of the FcRIIB on the BCR/ PKB response. It also suggests that binding of PI(3,4)P2 to the PH domain of PKB in vivo is insufficient to target PKB to the membrane. However, it should be emphasized that accumulation of PI(3,4)P2 in BCR/FcRIIb activated B cells and has not been formally shown.
The PKB-PH domain was recruited stably to the membrane in BCR-activated cells and also in cells expressing constitutively active PI3K. The PKB-PH domain binds to PIP3 and its translocation to the plasma membrane in B cells was absolutely dependent on the continued presence of D3 phosphoinositides: inhibition of the catalytic activity of PI3K with LY294002 causes immediate loss of the PKB-PH domain from the plasma membrane. It was particularly striking that within 30–40 s of adding the PI3K inhibitor, the PKB-PH domain moves from the plasma membrane into the cytosol and the nucleus of the B cell. The stability of the membrane localization of the PKB-PH domain was in marked contrast to the transient membrane residence of intact wild-type PKB under conditions of continuous PI3K activation or BCR triggering. The loss of wild-type active PKB from the plasma membrane within 1 min of BCR ligation is intriguing and cannot be explained by limits in levels of cellular PIP3 because PIP3 levels are elevated in A20 cells for at least 15 min after BCR ligation (data not shown). Moreover, PKB is not maintained at the membrane in cells expressing constitutively active PI3K although the stability of the membrane targeting of the PKB-PH domain confirms that there are sufficient levels of D-3 phosphoinositides in activated B cells to tether PKB to the membrane. Previous models have suggested that PKB colocalizes with active PI3K to membrane sites with elevated levels of D-3 phosphoinositides. The present confocal imaging data comparing the localization of full length PKB and the PKB-PH domain in B cells show that the PKB-PH domain follows this model but the full length PKB molecule does not. These studies thus reveal the existence of a molecular mechanism that must cause active PKB to dissociate from the plasma membrane in B cells despite the continued generation of PIP3. One possibility is that phosphorylated and/or active full length PKB undergoes a conformational change that prevents continued lipid binding. Alternatively, PKB interactions with substrate may relocalize the active kinase to the cytosol and nucleus.
In summary, this study has used confocal imaging coupled with more classical biochemical analyses to study the dynamic PI3K-regulated processes that occur during B cell activation. The BCR triggers a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of BCR-stimulated B cells. This result affords new information as to the potential site of action of PKB during B cell activation. Recent genetic studies have shown that PI3K is important for B cell development and for B cell function in the peripheral lymphoid compartment. The present results show that PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes which firmly positions Akt/PKB in PI3K-mediated signaling pathways in B lymphocytes. There has been much recent information about the effects of antigen receptors on membrane-localized signaling pathways in lymphocytes; the significance of the present report is that the PI3K/PKB pathway couples signaling events triggered at the lymphocyte membrane to the cell nucleus.
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Submitted: 2 April 1999
Revised: 21 May 1999
E. Astoul is supported by the European Community Training and Mobility of Researchers Program (ERBFMICT 960518).
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