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IMMUNOBIOLOGY
From the Department of Pediatrics, Beirne B. Carter
Center for Immunology Research, and Department of Microbiology,
University of Virginia, Charlottesville, VA.
Activation of the serine/threonine kinase Akt and the regulation of
its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B
lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is
attenuated by co-cross-linking of BCR with the inhibitory receptor
Fc In the early phase of the immune response, the
binding of the B-cell receptor (BCR) to antigen leads to receptor
cross-linking and activation. This results in intracellular calcium
flux, gene expression, protein synthesis, and cell
survival.1 Activation of phosphatidylinositol 3-kinase
(PI-3K) and the generation of the phospholipid phosphatidylinositol
3,4,5-trisphosphate (PIP3), are critical for optimal BCR-mediated
signaling.2 The rise in intracellular calcium, as well as
the activation of the serine/threonine kinase, protein kinase B (PKB),
or Akt (implicated in antiapoptotic cell survival signals), have been
shown to be downstream of PI-3 kinase.2-6 Thus, negative
regulation of PI-3 kinase-mediated signals is used as a mechanism for
down-modulation of B-cell responses (see below).
Akt/ PKB was identified originally as part of an oncogene
responsible for spontaneously arising leukemias and lymphomas in mice.7-11 Since then, many cytokines, growth factor
receptors, and antigen receptors have been shown to induce Akt kinase
activity.4,12,13 Akt activation has been linked to PI-3
kinase-dependent antiapoptotic signals delivered through these
receptors,14-18 as well as glucose metabolism,19-21 protein synthesis,21,22 and
gene expression.19,23-25 The human Akt locus has been
mapped to chromosome 14q32, proximal to the immunoglobulin heavy chain
locus.26 Because frequent translocations and inversions in
this region have been linked to T-cell leukemia/lymphoma and childhood
leukemia,8,27 elucidating the mechanism of Akt activation
and its regulation could have potential implications for tumorigenesis.
Akt contains an N-terminal pleckstrin homology (PH) domain that binds
the phospholipid PIP3, a glycine-rich region, followed by a kinase
domain and a C-terminal regulatory region.8,28 Generation
of PIP3 by PI-3 kinase in response to receptor activation results in
the Akt-PH domain binding to PIP3, and Akt recruitment to the
membrane.8,28,29 This membrane translocation is thought to
cause a conformational change in Akt, allowing its phosphorylation and
subsequent activation.13,29-32 The phosphorylation of Akt occurs on 2 specific sites, threonine 308 and serine 473, both of which
are required for maximal Akt activation.33 Phosphorylation of Akt on Thr308 is mediated by phosphoinositide-dependent
kinase1 (PDK1), whereas phosphorylation of Ser473 is
mediated by PDK2.34 Recent studies suggest that PDK2 may in fact be composed of PDK1 in complex with another
protein.35,36
PDK1 also contains a PH domain,37-40 and PDK1 recruitment
to the membrane also requires the binding of its PH domain to PIP3. It
appears that PDK1 can be constitutively localized on the membrane through the resting levels of PIP338; however, additional
PDK1 may be recruited to PIP3 generated on receptor
activation.41 In contrast, Akt is not constitutively on
the membrane and its membrane recruitment requires receptor
activation.8,34,38 Artificial membrane targeting of Akt
through the addition of a myristoylation sequence at its N-terminus,
leads to a constitutively active Akt and is sufficient for protection
of cells against apoptosis.15,16,18 In fact,
myristoylation of the gag sequence in v-Akt and its localization to the
membrane have been attributed to its oncogenic
potential.42 Thus, regulation of Akt membrane localization
and its activation status may be critical in regulating cellular
proliferation and oncogenesis.
In latter stages of the antibody responses, B lymphocytes are
negatively regulated by excess circulating antibody through simultaneous engagement of the BCR and the Fc Although the above studies clearly demonstrated a role for SHIP in
inhibiting Akt activation, the precise mechanism by which this
inhibition is achieved remains unclear. Several possibilities exist.
Destruction of PIP3 by enzymatic activity of SHIP could make PIP3
limiting for Akt PH domain binding, and thereby inhibit the membrane
localization of Akt. Alternatively, SHIP could negatively regulate PDK1
and indirectly mediate Akt inhibition. Both mechanisms may also be
operational concurrently. To better understand the SHIP-mediated
inhibition of Akt, we used an Akt that has been engineered to be
constitutively localized on the membrane through a myristoylation
motif, and fused to the hormone-binding domain of the murine estrogen
receptor (Myr Akt-ER).20 Despite being membrane bound,
activation of Myr Akt-ER is inducible only after the addition of the
drug 4-hydroxytamoxifen (4-HT). Because we observed that this Akt-ER
requires phosphorylation by PDKs, this allowed us to address whether
SHIP regulates Akt activation at the level of Akt, PDK or both. With
the use of stable B-cell lines expressing this Myr Akt-ER and by the
cross-linking of BCR with or without Fc Plasmids, cell lines, and antibodies
Transfections
Stimulations, immunoprecipitations, and immunoblotting Parental A20 cells or transfectants were washed and resuspended at 15 × 106 cells/mL in RPMI-1640. As indicated, cells were preincubated at 37°C with 1 µmol/L 4-hydroxy tamoxifen (4-HT) (Sigma) for 20 minutes to 3 hours.20 The cells were washed, resuspended in RPMI medium, and incubated for 5 minutes each with either 1.4 µg/mL antimouse F(ab')2 fragment to stimulate the BCR alone, or 2.5 µg/mL intact antimouse IgG for BCR+Fc RIIB1 co-cross-linking. Cells were quickly pelleted, and
lysed at 4°C for 15 minutes with lysis buffer, containing 50 mmol/L
Tris, pH 7.6, 150 mmol/L NaCl, 1% NP-40, 10 mmol/L Na pyrophosphate,
10 mmol/L NaF, 1 mmol/L Na3VO4, 2 mmol/L PMSF,
and 10 µg/mL each of aprotinin, leupeptin, pepstatin, and AEBSF.
After centrifugation at 14 000 rpm for 10 minutes at 4°C, the
supernatant was used directly for immunoblotting, or, specific proteins
were precipitated using the relevant antibody and protein A-conjugated
beads for 2 hours at 4°C. Beads were washed, and bound proteins were
analyzed using standard SDS-PAGE, and the immunoblots were developed
using chemiluminescence.
Kinase assays After 4-HT incubation and/or stimulation of BCR alone or BCR+FcRIIB1, 15 × 106 A20 lymphocytes were lysed
(same lysis buffer as above with addition of 1 µmol/L microcystin),
and Akt-ER was immunoprecipitated using anti-HA and protein A beads.
The Akt kinase assay using a GSK-3-derived substrate peptide was
performed using the Akt immunoprecipitation kinase assay kit purchased
from Upstate Biotechnology (Lake Placid, NY).4 The
p70S6k activity was measured under the same conditions
using 0.1 mmol/L Rsk substrate peptide (Santa Cruz Biotechnology).
Intracellular calcium measurements The 2 × 106 A20 lymphocytes were washed and resuspended in 1 mL RPMI-1640, and incubated in 1 µg/mL Indo 1 (Molecular Probes, Eugene, OR) for 30 minutes at 37°C. The cells were washed 3 times and resuspended in a buffer containing 10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 0.1% glucose, and 1% fetal calf serum. The calcium flux was recorded on excitation at 340 nm as the ratio of fluorescence emissions at 398 and 480 nm with an Hitachi 2000 spectrofluorimeter (Hitachi Instruments, Naperville, IL). The background was recorded for 30 seconds before addition of either 13 µg/mL of the antimouse F(ab')2 fragment for BCR cross-linking or 23 µg/mL of intact antimouse IgG for BCR+FcRIIB1
co-cross-linking. The data are presented as Indo-1 fluorescence ratio.
To determine the effect of Akt-ER activation on calcium flux, similar
recordings were performed after addition of 1 µmol/L 4-HT for 2 hours
to A20 cells before Indo-1 loading.
CTLL proliferation assay The 5 × 104 A20 cells in 200 µL/well in a 96-well plate were stimulated in triplicate with F(ab')2 or intact antimouse IgG antibody for 24 hours at different concentrations from 7.5 to 15 µg/mL. Where indicated, 1 µmol/L 4-HT was added and the drug was present throughout the 24-hour stimulation period. A 125 µL sample of the supernatants was collected from each well, frozen, and thawed once. IL-2-dependent CTLL cells (washed 4 times in phosphate-buffered saline [PBS] to remove any remaining IL-2 in the growth medium) were plated in 96-well plates at 104 per well in 50 µL fresh growth medium without IL-2. Supernatants from A20 cells (50 µL) was added to each well and incubated for 16 hours. 3H-thymidine was then added at 0.037 MBq (1 µCi) per well for an additional 5 hours, and the incorporated radioactivity was measured.55
Expression of a membrane-targeted, regulative Akt in B cells To study the regulation of Akt during BCR and FcRIIB1
signaling, murine A20 B lymphocytes were stably transfected with an inducible Akt construct previously used by Kohn et al20 to
study Akt activation in NIH3T3 cells and adipocytes. This modified Akt contains the catalytically active portion of the Akt kinase, but lacks
the PH domain necessary for membrane localization. Instead it is
targeted to the membrane through a myristoylation sequence added to its
amino terminus and was fused at the C-terminus to the hormone-binding
domain of murine estrogen receptor (ER) (Figure 1A). This myristoylated Akt-ER fusion
protein (Myr Akt-ER) remains inactive until the addition of the
estrogen analogue, 4-hydroxytamoxifen (4-HT),20 which is
thought to relieve steric inhibition of Akt by the ER domain. In the
presence of 4-HT, the Akt-ER protein becomes phosphorylated and this
has been correlated with Akt-ER activation.20 This
inducible Akt-ER provided a tool to address whether Akt inhibition by
FcRIIB1/SHIP is regulated at the level of membrane localization
and/or through the subsequent phosphorylation of Akt by PDKs. As a
control, we used an Akt-ER fusion protein that carried a mutation of
the critical +2 glycine within the myristoylation sequence and
therefore not targeted to the membrane (non-Myr Akt-ER) (Figure
1A).20
Multiple murine A20 clones stably expressing Myr Akt-ER and the Non-myr
Akt-ER were identified by anti-HA or anti-ER immunoprecipitations, followed by anti-HA immunoblotting (Figure 1B-C, respectively). The
functional data presented later in this manuscript were obtained from
several of these stable clones. BCR and Fc The Akt-ER can be activated in B cells by 4-HT Myr Akt-ER-expressing A20 B cells were incubated with 4-HT for varying periods to test the inducible activation. The cells were lysed and the Akt-ER was immunoprecipitated with anti-HA antibodies. The in vitro Akt kinase activity was determined using a glycogen synthase kinase-3 (GSK3)-derived peptide as a substrate (see "Materials and methods").4 In the absence of 4-HT, there was little Akt activity above background. On 4-HT addition, we observed a 20-fold increase in Akt activity within 30 minutes, and a continued linear increase in kinase activity after 3 hours (Figure 2A). This is consistent with the findings of Kohn et al.20 The influence of BCR on Akt-ER kinase activity was determined by cross-linking the cells with anti-BCR antibodies. Consistently, BCR cross-linking did not further enhance the Akt-ER kinase activity above the effect of 4-HT addition (Figures 2A and 3A). Although direct comparisons in kinase activities between the endogenous Akt and Akt-ER is not feasible because of 2 different precipitation antibodies being used, we consistently observed that the maximal Akt-ER activity (induced by 4-HT) is roughly comparable to the maximal endogenous Akt activity under similar kinase assay conditions (about a 30-fold increase in both cases). Non-Myr Akt-ER was not activated by tamoxifen addition above baseline in any of the conditions tested (Figure 3A and data not shown).
PDK1, the kinase that phosphorylates Akt, also contains a PH domain that binds PIP3.37-40 PDK1 and PDK2 (PDK1 in complex with another protein) have been shown to phosphorylate Akt on Thr308 and Ser473, respectively, in a PIP3-dependent manner. We tested whether Akt-ER activation would also be PIP3 dependent. Addition of wortmannin, a drug that inhibits PIP3 generation, significantly diminished the 4-HT-dependent Akt-ER kinase activation and this correlated with the inhibition of phosphorylation of Akt-ER on Ser473 and Thr308 (Figure 2B-C). We observed less inhibition of Thr308 phosphorylation, compared with Ser473, after wortmannin addition, although significance of this remains unclear. Membrane targeted Akt-ER is not inhibited by Fc RIIB1
and that this inhibition is dependent on SHIP. Here, we addressed
whether the Myr Akt-ER is sensitive to inhibition by FcRIIB1/SHIP.
Parental A20 cells, Myr Akt-ER, and non-Myr Akt-ER expressing cells
were pretreated with 4-HT and then stimulated by BCR cross-linking
alone, or BCR+FcRIIB1 co-cross-linking. The cells were lysed and
the Akt-ER kinase activities were measured after anti-HA immunoprecipitation.
Treatment of cells with 4-HT led to a marked increase in Myr Akt-ER
kinase activity (Figure 3A). This directly correlated with
PDK-dependent phosphorylation of Akt-ER from the same experiment determined by immunoblotting with antiphospho-Akt (Thr308)
antibody or the mobility shift in the Myr Akt-ER because of phosphorylation (Figure 3B-C). BCR cross-linking alone, in the absence
of tamoxifen, did not lead to phosphorylation of Akt-ER or to an
increase in kinase activity. Moreover, the kinase activity of Myr
Akt-ER induced by 4-HT was not further enhanced by BCR cross-linking
alone. Most importantly, BCR+Fc Although the above experiments clearly indicated that BCR-Fc Endogenous Akt, MAP kinase pathways and calcium flux are not affected in Akt-ER expressing cells Several signaling events are downstream of BCR-mediated PI-3 kinase induction and include the initiation of calcium flux2,45,56 and activation of MAP kinase (MAPK) (our unpublished data). Although these signaling pathways are all inhibited by the PI3-kinase inhibitor, wortmannin, they have not been shown to be contingent on the activation of Akt, or each other in B cells. We therefore tested whether 4-HT treatment of the cells containing the transfected Myr Akt-ER could influence these other PIP3-mediated events in a manner different from that in parental B cells.We first addressed the effect of Akt-ER expression on endogenous
Akt activation by BCR cross-linking and the inhibition because of
co-cross-linking with Fc
To examine the effect of BCR+Fc We also determined the pattern of BCR-induced intracellular calcium
flux and the inhibition of this flux by BCR+Fc The p70S6 kinase activation is independent of Akt activity in B cells and is not inhibited by SHIP The p70S6 kinase (p70S6k) has been recognized as a key downstream target of the PI-3 kinase signaling pathway.57 It has recently been demonstrated that PDK1 functions as an upstream kinase that phosphorylates both p70S6k and Akt.58 In some cases, an Akt-dependent phosphorylation of p70S6k has also been demonstrated.20,59,60 We examined the activation p70S6k in Myr Akt-ER-expressing cells in response to BCR activation and the effect of BCR+FcRIIB1
co-cross-linking. The cell lysates were immunoprecipitated with
anti-p70S6k antibody and the in vitro kinase assay was
carried out using a Rsk peptide substrate. As shown in Figure
5A, BCR alone cross-linking (in the
presence or absence of tamoxifen) led to a 5-fold increase in
endogenous p70S6k activity. Interestingly, BCR+FcRIIB1
co-cross-linking did not lead to an inhibition of
p70S6k activation.
Also, addition of 4-HT to these cells in the absence of BCR
cross-linking (ie, Akt activation alone), did not lead to a significant increase in p70S6k activity. Although this is in apparent
contrast to previous studies in which constitutive activation of Akt
led to an increase in p70S6k activity, this may be due to
differences in cell type.20,59 Moreover, we had examined
endogenous p70S6k, whereas the previous studies had
analyzed overexpressed p70S6k. BCR-dependent
p70S6k activation was still inhibited by wortmannin in
these cells, indicating that p70S6k activation still
occurred downstream of PI-3 kinase (data not shown). Consistent with
our observation that SHIP does not seem to regulate PDK-dependent
phosphorylation of Akt-ER, phosphorylation of p70S6k by
PDKs and the subsequent p70S6k activation was insensitive
to Fc Activation of membrane targeted Akt alone is insufficient to
overcome the Fc RIIB1.2,46,56 Since Eder et al61 have
demonstrated that Akt may play a role in IL-2 production in T cells, we
tested whether activated membrane-targeted Akt can influence downstream
events that lead to BCR-induced IL-2 production and whether it can
partially or fully "rescue" the FcRIIB1-mediated inhibition of
this IL-2 response. Parental A20 cells and those transfected with Myr
Akt-ER or non-Myr Akt-ER were stimulated by BCR cross-linking alone or
by BCR+ FcRIIB1, in the presence or absence of 4-HT, and the
secretion of IL-2 in the supernatants was tested (see "Materials and
methods").
As seen in Figure 6, all 3 cell types
produced IL-2 in response to BCR stimulation and this IL-2 secretion
was inhibited by BCR+Fc
Activation of the serine/threonine kinase Akt and the regulation
of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic
receptors.8,33,34,62 Previous studies have demonstrated
that the inositol phosphatase SHIP, through its dephosphorylation of
the membrane phospholipid PI(3,4,5)P3, inhibits Akt
activation.4-6 In this report, we addressed whether
SHIP-mediated destruction of PIP3 in B cells inhibits Akt directly at
the level of Akt recruitment to the membrane, at the level of PDK
recruitment/phosphorylation of Akt, or both. We demonstrate that a
constitutively membrane targeted, but inducible Akt construct is not
susceptible to Fc A number of studies have used transient, constitutive activation of Akt
through membrane targeting to demonstrate a role for Akt in protecting
cells against apoptosis induced by factor deprivation or/DNA
damage.16-18 However, one caveat with this approach is the presence of constitutively active protein throughout the time of the
experiment. The inducible Akt-ER designed by Kohn et al,20 and used in this report, remains completely inactive and allows the
tamoxifen-dependent activation of Akt when desired. The myristoylated Akt-ER fusion protein still required PDK-dependent phosphorylation for
activation, and required the prior addition of tamoxifen to "open
up" the Akt for phosphorylation by PDKs. Thus, this Akt-ER fusion
protein allowed us to delineate which step(s) of the Akt activation
sequence is inhibited by SHIP, by adding tamoxifen before, after, or
simultaneously with BCR stimulation or BCR+Fc Because the endogenous Akt in cells is recruited to the membrane during activation and has been shown to move to the nucleus after activation,5 whether the myristoylated Akt can also move to the nucleus has been debated.8 However, the v-Akt, which is also myristoylated (through the gag fusion), appears to be located at the membrane, cytosol, and in the nucleus and can mediate tumor formation.42 Similarly, engineered myristoylated Akt proteins have been shown to be functional and protect cells against apoptosis.16-18 Recently, Klippel et al60 have demonstrated that a similar myristoylated Akt-ER fusion protein can mediate cell cycle progression after tamoxifen addition. The rationale for our use of Myr Akt-ER was the specific advantage this construct provided in addressing SHIP-mediated regulation of Akt. The non-Myr Akt-ER was completely inactive even after tamoxifen addition and thus helped to rule out nonspecific effects due to stable expression of Akt-ER fusion proteins in the B cells. Several lines of evidence shown here suggested that SHIP regulates Akt
primarily at the level of Akt recruitment to the membrane. First, once
activated by tamoxifen addition, the Akt-ER was resistant to inhibition
by Fc We examined the possibility that Fc It is noteworthy that even though both calcium flux and Akt activation
are PIP3-dependent events in B cells, BCR-induced calcium flux and its
inhibition by BCR+Fc Although not a normal feature of B cells, activation via the BCR in the
A20 B cells leads to IL-2 gene transcription and the secretion of IL-2.
It has been previously reported that in T cells, Akt may play a role in
regulating IL-2 gene transcription.61 However, we failed
to observe an increase in IL-2 production in Myr Akt-ER expressing
cells after BCR activation in the presence of tamoxifen. Additionally,
we did not see a rescue as a result of Akt-ER of the inhibition of IL-2
production by Fc We also observed that a putative Akt cytosolic target,
p70S6k, appeared to be independent of Akt membrane
localization and activation. Although this is in contrast to previously
published reports,20,59,60 the difference could be
attributed to cell type or to the fact that we examined endogenous
p70S6k activation, whereas the previous studies addressed
activation of transiently overexpressed p70S6k.
Interestingly, although p70S6k was activated 5-fold by BCR
stimulation, co-cross-linking of BCR+Fc In summary, we have examined the regulation of Akt membrane
translocation under inhibitory signaling conditions in B cells and
demonstrate that Akt inhibition by Fc
We thank Dr Richard Roth for providing the plasmids encoding Myr Akt-ER and non-Myr Akt-ER, and we also thank the members of the Ravichandran laboratory for their helpful discussions.
Submitted November 5, 1999; accepted April 13, 2000.
Supported by a grant from the National Institutes of Health (AI 43425) to K.S.R.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kodimangalam S. Ravichandran, Beirne B. Carter Center, Bldg MR4, Rm 4012F, HSC, University of Virginia, Charlottesville, VA 22908; email: kr4h{at}virginia.edu.
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Top
Abstract
Introduction
4-129Akt-ER construct and
the control construct with an A2 mutation in the myristoylation
sequence subcloned into the PWZLneo plasmid were generously provided by Dr Richard Roth (Stanford, CA).
-mercaptoethanol (Sigma, St Louis, MO). The CTLL-2 line was grown in
the same media with 5 U/mL of recombinant interleukin-2 (IL-2)
(Hoffman-LaRoche, Nutley, NJ). Rabbit antimouse whole Ig antibody and
the F(ab')2 fragment were obtained from Jackson
Immunoresearch Labs (West Grove, PA). The anti-HA 12CA5 murine
monoclonal antibody and the 2.4G2 rat monoclonal antibody against
Fc
, the donkey
antimouse IgG, FITC-labeled antidonkey secondary antibody, and the
mouse monoclonal antibody specific for the carboxy-terminus of
p70S6k were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Phosphospecific antibodies to Thr308 or
Ser473 of murine Akt and the antibody to phopsho-p44 MAPK
were from New England Biolabs (Beverly, MA).
A mutation
within this sequence (in the non-Myr Akt-ER version) was added to the
N-terminus of PH-domain deleted (region between aa 4-129) Akt. This
Akt, followed by a hemagglutinin (HA) tag, was fused to the estrogen
receptor hormone-binding domain (ER). (B) A20 cells were transfected
with Akt-ER constructs and the expression in different clones of Myr
Akt-ER (clones 8, 24, 51, 58) or non-Myr Akt-ER (clones 1M, 13M) was
analyzed by anti-HA immunoprecipitation, followed by anti-HA
immunoblotting. (C) Lysates from some of the clones were
immunoprecipitated with anti-ER antibodies and immunoblotted with
anti-HA. Molecular mass standards (in kilodaltons) are indicated to the
left. (D) Comparable expression of BCR and Fc
