Phosphatidylinositol 3-Kinase Kinase-3 Signaling Pathway Via Akt

The B Cell Antigen Receptor Activates the Akt (Protein Kinase B)/Glycogen Synthase Kinase-3 Signaling Pathway Via Phosphatidylinositol 3-Kinase This information is current as of September 28, 2021. Michael R. Gold, Michael P. Scheid, Lorna Santos, May Dang-Lawson, Richard A. Roth, Linda Matsuuchi, Vincent Duronio and Danielle L. Krebs J Immunol 1999; 163:1894-1905; ; http://www.jimmunol.org/content/163/4/1894 Downloaded from

References This article cites 82 articles, 45 of which you can access for free at:

http://www.jimmunol.org/content/163/4/1894.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The B Cell Antigen Receptor Activates the Akt (Protein Kinase B)/Glycogen Synthase Kinase-3 Signaling Pathway Via Phosphatidylinositol 3-Kinase1

Michael R. Gold,2* Michael P. Scheid,† Lorna Santos,‡ May Dang-Lawson,* Richard A. Roth,§ Linda Matsuuchi,‡ Vincent Duronio,† and Danielle L. Krebs3*

We have previously shown that the B cell Ag receptor (BCR) activates phosphatidylinositol (PI) 3-kinase. We now show that a serine/threonine kinase called Akt or protein kinase B is a downstream target of PI 3-kinase in B cells. Akt has been shown to promote cell survival as well as the transcription and translation of proteins involved in cell cycle progression. Using an Ab that speciﬁcally recognizes the activated form of Akt that is phosphorylated on serine 473, we show that BCR engagement activates Akt

in a PI 3-kinase-dependent manner. These results were confirmed using in vitro kinase assays. Moreover, BCR ligation also Downloaded from induced phosphorylation of Akt of threonine 308, another modification that is required for activation of Akt. In the DT40 chicken B cell line, phosphorylation of Akt on serine 473 was completely dependent on the Lyn tyrosine kinase, while the Syk tyrosine kinase was required for sustained phosphorylation of Akt. Complementary experiments in BCR-expressing AtT20 endocrine cells confirmed that Src kinases are sufficient for BCR-induced Akt phosphorylation, but that Syk is required for sustained phosphorylation of Akt on both serine 473 and threonine 308. In insulin-responsive cells, Akt phosphorylates and inactivates the serine/ threonine kinase glycogen synthase kinase-3 (GSK-3). Inactivation of GSK-3 may promote nuclear accumulation of several http://www.jimmunol.org/ transcription factors, including NF-ATc. We found that BCR engagement induced GSK-3 phosphorylation and decreased GSK-3 enzyme activity. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling pathway. The Journal of Immunology, 1999, 163: 1894–1905.

ngagement of the B cell Ag receptor (BCR)4 by Ags ini- line (4, 5), until recently little was known about the downstream tiates signals that can promote apoptosis, anergy, survival, targets of PI 3-kinase signaling in B cells. E activation, or proliferation, depending upon the differen- Recent work has revealed that PI 3-kinase plays a key role in tiation state of the B cell, the magnitude, and duration of the BCR multiple cellular processes. PI 3,4,5-trisphosphate (PIP3), a plasma by guest on September 28, 2021 signal, and whether or not the B cell receives additional signals membrane phospholipid produced by PI 3-kinase, is a ligand for from CD40 or cytokine receptors (1, 2). When clustered by mul- the pleckstrin homology (PH) domains that are found in a variety tivalent Ags or by anti-Ig Abs, the BCR activates three key sig- of cytosolic proteins, many of which are involved in signal trans- naling enzymes: Ras, phospholipase C-␥ (PLC-␥), and phosphati- duction (6). By binding PH domains, PIP3 can recruit PH domain- dylinositol (PI) 3-kinase (1, 3). Although PI 3-kinase had been containing proteins to the plasma membrane. This is of particular shown to contribute to the ability of the BCR to regulate the pro- importance for cytosolic signaling enzymes that act on membrane- liferation of normal B cells, as well as a human B lymphoma cell associated substrates. In addition to recruiting signaling proteins to

the plasma membrane, the binding of PIP3 to a protein’s PH domain may induce conformational changes that affect its enzymatic

Departments of *Microbiology and Immunology, †Medicine, and ‡Zoology, Univer- activity, its ability to act as a substrate for kinases, or its ability to sity of British Columbia, Vancouver, British Columbia, Canada; and §Department of interact with other proteins. Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA In B cells, PIP -PH domain interactions play an essential role in 94305 3 the ability of the BCR to stimulate PLC-␥-dependent signaling. Received for publication February 18, 1999. Accepted for publication June 9, 1999. The Btk tyrosine kinase, which phosphorylates and activates The costs of publication of this article were defrayed in part by the payment of page ␥ charges. This article must therefore be hereby marked advertisement in accordance PLC- , is recruited to the plasma membrane via the binding of its with 18 U.S.C. Section 1734 solely to indicate this fact. PH domain to PIP3 (7–10). PIP3-PH domain interactions are also 1 This work was supported by grants from the Medical Research Council of Canada involved in activation of the Rac1 GTPase, which is required for (to M.R.G., L.M., and V.D), a grant from the Natural Sciences and Engineering sustained hydrolysis of PI 4,5-bisphosphate by PLC-␥ (11). Rac1- Research Council of Canada (to M.R.G.), Medical Research Council Scholarships (to M.R.G. and V.D.), a Cancer Research Society (Canada) studentship (to M.P.S.), and GTP activates PI 4-phosphate 5-kinases, which are responsible for a University of British Columbia Graduate Fellowship (to D.L.K.). providing a continuous supply of PI 4,5-bisphosphate to act as a 2 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of substrate for PLC-␥ (12–14). Both Sos and Vav can act as ex- Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: change factors that activate Rac1, and their ability to do so is [email protected] dependent on the binding of PIP3 to their PH domains (15, 16). In 3 Current address: Walter and Eliza Hall Institute for Medical Research, Melbourne, addition to Btk, SOS, and Vav, a large number of other PH domain- Victoria 3050, Australia. containing proteins have been identiﬁed, many of them cytosolic sig- 4 Abbreviations used in this paper: BCR, B cell Ag receptor; PLC-␥, phospholipase naling proteins (6). Thus, it is likely that the activation of PI 3-kinase ␥ C- , PI, phosphatidylinositol; PIP3, PI 3,4,5-trisphosphate; PH, pleckstrin homology; by the BCR and the subsequent production of PIP3 regulates many PI(3,4)P2, PI 3,4-bisphosphate; ILK, integrin-linked kinase; GSK-3, glycogen synthase kinase-3; HA, hemagglutinin. other signaling pathways in addition to the PLC-␥ pathway.

There is now considerable evidence that a 60-kDa serine/thre- forms of Akt also selectively enhance the translation of cyclin D1 onine protein kinase called Akt (or protein kinase B) is a major (47). Thus, Akt may promote cell cycle progression at both the downstream target of PI 3-kinase signaling. Akt is activated by a transcriptional and posttranscriptional levels. number of receptors that activate PI 3-kinase, including the recep- Another major downstream target of Akt is glycogen synthase tors for insulin, platelet-derived growth factor, epidermal growth kinase-3 (GSK-3), a constitutively active serine/threonine kinase factor, IL-2, IL-3, IL-4, GM-CSF, and stem cell factor (17–24). In whose activity is inhibited by Akt. Regulation of GSK-3 activity these systems, it has been clearly shown that activation of PI 3-ki- has been studied primarily in the context of insulin receptor sig- nase is both necessary and sufficient for Akt to be activated. Re- naling. Insulin receptor signaling leads to a reduction in GSK-3 ceptor-induced activation of Akt is blocked by PI 3-kinase inhib- activity (20, 48, 49), and this appears to be mediated by Akt, since itors and by expression of dominant negative forms of PI 3-kinase a dominant-negative form of Akt can block the ability of insulin to (17–24). Conversely, expression of constitutively active forms of decrease GSK-3 activity (48). In vitro, Akt can phosphorylate PI 3-kinase in cells results in activation of Akt (25). GSK-3␣ on serine 21 and GSK-3␤ on serine 9, and these modi- Considerable progress has been made toward understanding fications correlate with inhibition of GSK-3 enzyme activity (20). how PI 3-kinase activates Akt (reviewed in Ref. 26). Akt has a PH Thus, GSK-3␣ and GSK-3␤ may be physiological substrates domain that binds the PI 3-kinase-derived lipids PIP and PI 3,4- 3 of Akt. bisphosphate (PI(3,4)P ) (27). This interaction recruits Akt to the 2 GSK-3-mediated phosphorylation may regulate a number of dif- plasma membrane and induces a conformational change that al- ferent cellular processes. First, GSK-3 may regulate the subcellular lows it to be phosphorylated and activated by upstream kinases localization of the NF-ATc transcription factor. The BCR and (28). In response to receptor engagement, human Akt is phosphor- Downloaded from other receptors that cause increases in intracellular Ca2ϩ concen- ylated on threonine 308 and serine 473. Phosphorylation of both of trations activate the Ca2ϩ-dependent phosphatase calcineurin, these residues is required for maximal activation of Akt. Changing which dephosphorylates NF-AT (50). This reveals a nuclear threonine 308 to an alanine completely blocks activation of Akt, c localization signal that allows NF-AT to translocate into the while changing serine 473 to an alanine significantly compromises c Akt activation (19, 29, 30). A kinase called PDK1 has been iso- nucleus, bind to AP-1-like transcription factors, and stimulate transcription. GSK-3 opposes this process by phosphorylating lated that phosphorylates threonine 308, but not serine 473 (31). http://www.jimmunol.org/ NF-ATc. This causes a conformational change that reveals a nu- PDK1 also contains a PH domain, and the production of PIP3 by PI 3-kinase is required to recruit PDK1 to the plasma membrane so clear export signal, resulting in NF-ATc being rapidly exported that it can phosphorylate Akt (32). The kinase that phosphorylates from the nucleus (51, 52). Thus, Akt-dependent inhibition of serine 473 of Akt has been termed PDK2. Although it has yet to be GSK-3 activity may be required for NF-ATc to accumulate in the identified definitively, there is some evidence that the integrin- nucleus and promote transcription. A number of other potential linked kinase (ILK) can phosphorylate this site on Akt (33). GSK-3 substrates have been identified (53), including the micro- Akt is a multifunctional mediator of PI 3-kinase-dependent sig- tubule-associated protein tau (54), the ⑀ subunit of the eIF-2B naling. First, PI 3-kinase activity is essential for the prevention of translation initiation factor (55), and ␤-catenin (56). Phosphoryla- apoptosis in a number of cell types (34–36) and this appears to be tion of ␤-catenin promotes its degradation (57). Inhibition of by guest on September 28, 2021 mediated by Akt (reviewed in Refs. 26, 37, and 38). Expression of GSK-3 by Wnt family receptors has been shown to play an im- a constitutively active form of Akt is sufficient to protect cells from portant role in development by stabilizing ␤-catenin and allowing apoptosis caused by growth factor withdrawal, while dominant- ␤-catenin/LEF-1 transcription factor complexes to accumulate in negative forms of Akt can cause apoptosis (36, 39). Several groups the nucleus (56). Recent work has also suggested that GSK-3- have proposed that Akt prevents apoptosis by phosphorylating Bad mediated phosphorylation of yet unidentified substrates can pro- (22, 40), a death-promoting member of the Bcl-2 family. This re- mote apoptosis and that Akt-dependent inhibition of GSK-3 activ- mains controversial, however, since other kinases can apparently ity promotes cell survival (58). Thus, like Akt, GSK-3 may be phosphorylate Bad (24) and the physiological relevance of Bad involved in a variety of important cellular functions, including phosphorylation has yet to be established. Thus, there are likely to transcription, translation, and cell survival. be additional mechanisms by which Akt can prevent apoptosis. Although BCR engagement activates PI 3-kinase (4) and causes Consistent with this idea, Cardone et al. (41) showed that Akt can a significant increase in the levels of PI 3-kinase-derived lipids in phosphorylate caspase-9 and prevent its proteolytic activation. Re- B cells (59), the effects of BCR signaling on Akt and GSK-3 have cent work has shown that Akt may also prevent apoptosis by phos- not been examined. Since Akt and GSK-3 may regulate important phorylating forkhead family transcription factors (42). Phosphor- cell functions, we asked whether these enzymes were targets of ylation of these transcription factors causes them to be exported BCR signaling. In this report, we show that both Akt and GSK-3 from the nucleus and prevents them from inducing the expression are regulated by the BCR in a PI 3-kinase-dependent manner. of proapoptotic genes, such as the Fas ligand. Thus, BCR ligation initiates a PI 3-kinase/Akt/GSK-3 signaling Another important function of Akt may be to promote cell cycle pathway. progression. Expressing a constitutively active form of Akt in a T cell line has been shown to stimulate the activity of E2F, a transcription factor that induces the expression of cyclin D3 and other Materials and Methods genes required for the G1 to S transition (43). Consistent with a role for Akt in cell growth and proliferation, Akt may also increase Materials the rate of protein synthesis by either directly or indirectly stim- Goat Abs specific for mouse IgM, mouse IgG, and human IgM were ob- ulating phosphorylation of the p70S6K kinase (21) and 4E-BP1 tained from Bio-Can (Mississauga, Ontario, Canada). Goat Abs against (44). p70S6K phosphorylates the ribosomal S6 protein, and this murine ␬ light chain were from Southern Biotechnology Associates (Bir- preferentially increases the translation of mRNAs containing 5Ј- mingham, AL). The 6E4 mAb to chicken Ig light chain (60) was a gift of M. Ratcliffe (McGill University, Montreal, Quebec, Canada). Wortmannin terminal oligopolypyrimidine tracts (45). Phosphorylation of 4E- and LY294002 were from Biomol (Plymouth Meeting, PA). Microcys- BP1 releases the eIF-4E translation initiation factor and allows it to tin-LR was obtained from Alexis (San Diego, CA). Avidin-conjugated aga- promote translation (46). Recent work has shown that activated rose was from Pierce (Rockford, IL). 1896 REGULATION OF Akt AND GSK-3 BY THE BCR

B cell lines and murine splenic B cells (Syk13 cells) have been described previously (66, 67). These cells were cultured in DMEM supplemented with 10% FCS and grown to near con- The WEHI-231 and A20 murine B cell lines, as well as the RAMOS human fluence in 10-cm tissue culture dishes. Before being stimulated, the cells B cell line, were grown in RPMI 1640 supplemented with 10% heat-inac- were washed with PBS and cultured overnight under low serum conditions ␮ tivated FCS (Intergen, Purchase, NY), 50 M 2-ME, 1 mM pyruvate, and (DMEM with 0.2% FCS). The cells were then washed with PBS and in- 2 mM glutamine. To reduce the signaling contribution from serum-derived cubated for 15 min at 37°C in modified HEPES-buffered saline (see above) growth factors, A20 cells (and in some experiments, WEHI-231 cells) were to further reduce any signaling due to serum growth factors. The cells were washed twice with RPMI 1640 and then cultured for 12–18 h at 37°C in washed again with PBS, and 10 ml of 37°C modified HEPES-buffered serum-free medium (RPMI 1640 supplemented with 0.5 mg/ml BSA, 50 saline was added to each dish. BCR signaling was initiated by adding goat ␮ M 2-ME, 1 mM pyruvate, and 2 mM glutamine) before being stimulated. anti-mouse IgM Abs to a final concentration of 20 ␮g/ml. Reactions were The wild-type DT40 chicken B cell line, as well as the Lyn-deficient (61), terminated by aspirating the medium, washing the cells twice with ice-cold Syk-deficient (61), Btk-deficient (62), and PLC-␥2-deficient (63) variants PBS containing 1 mM Na3VO4, and then solubilizing the cells with 1 ml of this cell line, were grown in RPMI 1640 supplemented with 10% heat- buffer A (see above). After 20 min on a rocker in the cold, the cell lysate inactivated FCS, 1% heat-inactivated chicken serum (Life Technologies, was collected, detergent-insoluble material was removed by centrifugation, ␮ Grand Island, NY), 50 M 2-ME, and 4 mM glutamine. Small resting B and protein concentrations were determined using the BCA assay. cells were isolated from the spleens of C57BL/6 mice by Percoll density centrifugation after Ab- and complement-mediated lysis of T cells. Briefly, Immunoblotting spleen cells were incubated at 37°C for 45 min with a 1:4 dilution of guinea pig complement (Life Technologies) and 1:4 dilutions of culture superna- Cell lysates (60 ␮g protein) were separated on 10% SDS-PAGE gels and tants from the HO13.4 anti-Thy1 hybridoma (American Type Culture Col- transferred to nitrocellulose membranes. For detection of phosphorylated lection (ATCC), Manassas, VA), the 3.155 anti-CD8 hybridoma (ATCC), Akt, the membranes were blocked for2hatroom temperature with 5% and the 2B6 2D8 anti-CD4 hybridoma (from D. Hanson, Washington Uni- (w/v) nonfat dry milk powder in TBST (10 mM Tris-HCl (pH 7.5), 150

versity, St. Louis, MO). The small resting B cells were recovered from the mM NaCl, 0.1% Tween 20). The filters were then rinsed three times with Downloaded from interface of 60% and 75% isotonic Percoll layers. Flow cytometry showed TBST before being incubated overnight in the cold with anti-phospho-Akt that the resulting population of cells was Ͼ90% IgMϩ B cells. Abs (New England Biolabs, Mississauga, Ontario, Canada), which were diluted 1:1000 in TBST plus 5% BSA. One Ab specifically recognizes Akt Expression of epitope-tagged Akt in WEHI-231 cells that is phosphorylated on serine 473, while the other Ab specifically rec- Akt cDNA was cloned by PCR using HeLa cell cDNA as a template and ognizes Akt that is phosphorylated on threonine 308. After the overnight primers that added the influenza hemagglutinin (HA) epitope tag at the C incubation, the filters were washed three times for 10 min with TBST and terminus (18). The HA-Akt was originally cloned into the pECE expression then incubated for1hatroom temperature with HRP-conjugated goat http://www.jimmunol.org/ vector. For expressing the HA-Akt in WEHI-231 cells, we subcloned the anti-mouse IgG Abs (Bio-Rad, Richmond, CA) diluted 1:20,000 in TBST HA-Akt cDNA into the pLXSN retroviral expression vector. The HA-Akt plus 5% nonfat dry milk powder. The membranes were washed extensively was excised from pECE by digesting with EcoRI and BamHI and then with TBST, and immunoreactive bands were visualized by enhanced ligated into pLXSN that had been digested with the same enzymes. The chemiluminescence detection (Amersham Pharmacia Biotech). To reprobe resulting HA-Akt-pLXSN plasmid was transfected into the BOSC23 pack- these blots, bound Abs were eluted by incubating the blots for 1 h with 10 aging cell line (64) using the calcium phosphate precipitation method, and mM Tris-HCl (pH 1.6)/150 mM NaCl. The membranes were blocked, as the resulting retrovirus particles were used to infect WEHI-231 cells. A described above, and then incubated overnight in the cold with an anti-Akt detailed protocol for retrovirus-mediated gene transfer into WEHI-231 Ab (New England Biolabs) diluted 1:1250 in TBST plus 5% BSA. cells has recently been described by Krebs et al. (65). Two days postin- Immunoreactive bands were visualized as described for the anti- fection, the WEHI-231 cells were transferred into medium containing 1.8 phospho-Akt Abs. Detection of phosphorylated GSK-3␤ by immunoblotting was per- mg/ml G418 (Life Technologies) and cloned by limiting dilution. G418- by guest on September 28, 2021 resistant colonies were expanded and HA-Akt expression was assessed by formed in a similar manner with the following three exceptions. First, the immunoblotting with a biotinylated form of the 12CA5 anti-HA mAb concentration of Tween 20 in the TBST was reduced to 0.05% for all buffers. Second, the primary Ab was a rabbit polyclonal Ab that specifi- (Boehringer Mannheim, Laval, Quebec, Canada), followed by HRP-con- ␤ jugated streptavidin (Amersham Pharmacia Biotech, Piscataway, NJ). cally recognizes GSK-3 that is phosphorylated on serine 9 (Chemicon, Temecula, CA). This Ab was diluted 1:300 in TBST. Finally, the blots B cell stimulation and preparation of cell lysates were reprobed with an anti-GSK-3␤ mAb (Chemicon; diluted 1:1000 in TBST), which was visualized using HRP-conjugated sheep anti-mouse IgG The cells were washed once with modified HEPES-buffered saline (25 mM (Amersham; diluted 1:10,000 in TBST) and enhanced chemiluminescence sodium HEPES (pH 7.2), 125 mM NaCl, 5 mM KCl, 1 mM CaCl2,1mM detection. Na2HPO4, 0.5 mM MgSO4, 1 mg/ml glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 ␮M 2-ME) and then resuspended in this buffer at Akt in vitro kinase assays 107/ml for tissue culture cells or 2.5 ϫ 107/ml for splenic B cells. The cells were warmed to 37°C for 30 min and then stimulated with anti-Ig Abs. In Akt in vitro kinase assays were performed as described by Scheid and some experiments, the cells were incubated with the PI 3-kinase inhibitors Duronio (24). Akt was immunoprecipitated from cell lysates using 2 ␮gof wortmannin or LY294002 for 20 min at 37°C before the addition of anti-Ig anti-PKB-␣ Ab (Upstate Biotechnology, Lake Placid, NY; catalogue no. Abs. Reactions were terminated by adding ice-cold PBS containing 1 mM 06-558) that had been prebound to 20 ␮l of protein G-Sepharose beads (Sigma, St. Louis, MO). After mixing the cell lysates with the immobilized Na3VO4 and then centrifuging the cells in the cold for 15 s at full speed in a microfuge. The cell pellets were washed once, without resuspending, anti-PKB-␣ Absfor1hat4°C, the beads were washed once with fresh buffer B, three times with buffer B containing 500 mM NaCl, and once with with cold PBS/Na3VO4 and then solubilized. For immunoblotting or immunoprecipitation experiments, the cell pellets were solubilized in buffer A Akt kinase reaction buffer (20 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM ␮ (20 mM Tris-HCl (pH 8), 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, Na3VO4, 1 mM DTT, 0.25 mM PMSF, 1 g/ml microcystin-LR). The 10% glycerol, 1 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 1 mM beads were then resuspended in 20 ␮l of Akt kinase reaction buffer con- ␤ taining 60 ␮M Crosstide (Upstate Biotechnology) as a substrate. Reactions Na3VO4,25mM -glycerophosphate). After 10 min on ice, detergent- insoluble material was removed by centrifugation, and protein concentra- were initiated by adding 10 ␮l of Akt kinase reaction buffer containing 500 ␮ ␮ ␥ 32 tions for the detergent-soluble supernatant fractions were determined using M ATP, 75 mM MgCl2, and 10 Ci [ - P]ATP. After 15 min at 30°C, the bicinchoninic acid assay (Pierce). These samples were stored at Ϫ80°C 20 ␮l of the reaction mixture was spotted onto a 2 ϫ 2-cm square of until analyzed. For in vitro kinase assays, the cell pellets were solubilized Whatman (Tewksbury, MA) p81 phosphocellulose chromatography paper. in buffer B (50 mM Tris-HCl (pH 7.7), 0.5% Nonidet P-40, 2.5 mM EDTA, The filter papers were washed extensively with 1% phosphoric acid to ␤ remove free [␥-32P]ATP. Radioactivity that remained bound to the filters 20 mM -glycerophosphate, 10 mM NaF, 1 mM Na2MoO4,1mM ␮ ␮ ␮ ␮ was quantitated by liquid scintillation counting in the presence of scintil- Na3VO4, 0.25 M PMSF, 1 M pepstatin, 0.5 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, 1 ␮g/ml microcystin-LR), detergent-insoluble lation fluor. material was removed by centrifugation, and the detergent-soluble supernatant fractions were used immediately for in vitro kinase assays. GSK-3␣ in vitro kinase assays Growth and stimulation of AtT20 cells Cell lysates were mixed with 2 ␮g of anti-GSK-3␣ Ab (Upstate Biotech- nology) for1hat4°C. Immune complexes were recovered by adding 20 Transfected variants of the AtT20 endocrine cell line that express the com- ␮l of protein G-Sepharose beads and mixing for an additional hour. The plete BCR (100.33 cells) or the complete BCR plus the Syk tyrosine kinase beads were then washed four times with fresh buffer B and once with The Journal of Immunology 1897

GSK-3 kinase reaction buffer (5 mM HEPES (pH 7.4), 0.5 mM EDTA, 1 ␮ ␮ mM Na3VO4,1 g/ml microcystin-LR) before being resuspended in 30 l GSK-3 kinase reaction buffer containing 75 ␮M phosphoglycogen synthase peptide-2 (Upstate Biotechnology). Reactions were initiated by adding 10 ␮l of GSK-3 kinase reaction buffer containing 500 ␮M ATP, 75 mM ␮ ␥ 32 ␮ MgCl2 and 5 Ci [ - P]ATP. After 15 min at 30°C, 25 l of the reaction mixture was spotted onto a 2 ϫ 2-cm square of Whatman p81 chromatography paper. The ﬁlter papers were washed with 1% phosphoric acid and counted.

Results BCR-induced phosphorylation of Akt on serine 473 Phosphorylation of Akt on threonine 308 and serine 473 are essential for its activation (19, 29, 30). Thus, immunoblotting with an anti-peptide Ab that specifically recognizes Akt that is phosphorylated on serine 473 can be used as an indirect measure of Akt activation (68, 69). Such phosphorylation state-specific Abs are now commonly used to assess the activation state of a number of Downloaded from kinases that are activated by phosphorylation, including the ERK, JNK, and p38 mitogen-activated protein kinases. To determine whether BCR engagement might activate Akt, we stimulated B cell lines with anti-Ig Abs for various times and then performed anti- phospho-Akt immunoblots. Fig. 1, A–D, shows that Akt was not phosphorylated to a significant extent in unstimulated cells, but http://www.jimmunol.org/ that BCR ligation caused a substantial increase in phosphorylation of Akt on serine 473. This response was observed in the immature murine B cell line WEHI-231, the IgGϩ murine B cell line A20, the RAMOS human B cell line, and the DT40 chicken B cell line. In WEHI-231 cells, phosphorylation of Akt peaked at 1–5 min and then declined to lower levels at 15–60 min (Fig. 1A). In contrast, BCR-induced phosphorylation of Akt was sustained at high levels for 30–60 min in the A20, RAMOS, and DT40 cell lines. BCR engagement also stimulated phosphorylation of Akt on serine 473 by guest on September 28, 2021 in mature resting B cells isolated from mouse spleen (Fig. 1E). In anti-IgM-stimulated splenic B cells, Akt phosphorylation was very strong at 1.5–15 min and then declined to lower levels at 30–60 min. Although there were differences in the duration of the response, our results clearly show that Akt phosphorylation is a consistent characteristic of BCR signaling. To confirm that the protein being recognized by the anti- phospho-Akt Ab was in fact Akt, as opposed to an unrelated comi- grating protein, we expressed an epitope-tagged version of Akt in WEHI-231 cells. Anti-Akt immunoblotting of total cell lysates from this stable WEHI-231 clone showed that the HA-tagged Akt was expressed at similar levels as the endogenous Akt protein (Fig. 2, lower panel). The HA tagged-Akt was then immunoprecipitated using the 12CA5 anti-HA mAb and analyzed by sequential immunoblotting with the anti-phospho-Akt Ab and the anti-Akt Ab. Fig. FIGURE 1. BCR ligation induces phosphorylation of Akt on serine 473. A, WEHI-231 cells were stimulated with 40 ␮g/ml goat anti-mouse IgM 2 shows that BCR ligation induced phosphorylation of the HA-Akt Abs; B, A20 cells were stimulated with 40 ␮g/ml goat anti-mouse IgG Abs; on serine 473. The anti-HA-precipitated protein that was recog- C, RAMOS cells were stimulated with 40 ␮g/ml goat anti-human IgM Abs; nized by both the anti-phospho-Akt Ab and the anti-Akt Ab had and D, DT40 cells were stimulated with 20 ␮g/ml of the 6E4 anti-chicken the same electrophoretic mobility as the HA-Akt present in the cell Ig light chain mAb (anti-L chain) for the indicated times. E, Small resting lysates. This result confirms that BCR engagement leads to phos- B cells from mouse spleen were stimulated with 50 ␮g/ml goat anti-mouse phorylation of Akt at a site that plays a role in Akt activation. It is IgM Abs for the indicated times. Cell lysates (60 ␮g protein) were sepa- worth noting that, in response to BCR ligation, the HA-tagged Akt rated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the Ab that specifically recognizes Akt that is phosphory- was phosphorylated on serine 473 to a much lesser extent than the lated on serine 473 (upper panels). The blots were then stripped and endogenous Akt, even though they were expressed at similar lev- reprobed with an Ab that recognizes both phosphorylated and nonphos- els. The C-terminal HA tag is very close to serine 473 (18) and phorylated Akt (lower panels). The positions of the phosphorylated Akt may interfere with either the ability of PDK2 to phosphorylate Akt (P-Ser473-Akt), as well as the total Akt population (Akt), are indicated by on this residue or with the ability of the anti-phospho-Akt Ab to arrows. Molecular mass standards (in kDa) are indicated to the left of each bind to this site. Thus, in all additional experiments, we analyzed panel. Each experiment was performed at least twice with similar results. only the endogenous Akt protein. 1898 REGULATION OF Akt AND GSK-3 BY THE BCR

FIGURE 2. BCR ligation induces phosphorylation of a transfected HA- tagged Akt on serine 473. WEHI-231 cells expressing HA-tagged Akt were stimulated with 40 ␮g/ml goat anti-mouse IgM Abs for the indicated times. The HA-Akt was immunoprecipitated from cell lysates using the a biotinylated form of the 12CA5 anti-HA mAb plus avidin-conjugated agarose. As a control (C), lysate from anti-IgM-stimulated cells was incubated with avidin-agarose beads in the absence of the 12CA5 mAb. The precipitated proteins as well as total cell lysates (60 ␮g protein) from the same cells were separated on the same SDS-PAGE gel, transferred to nitrocellulose, FIGURE 3. Wortmannin blocks BCR-induced phosphorylation of Akt and analyzed by immunoblotting with the Ab that recognizes Akt that is serine 473. A, Splenic B cells were incubated for 20 min at 37°C with (ϩ) Downloaded from phosphorylated on serine 473 (upper panel). The blots were then stripped or without (Ϫ) 30 nM wortmannin before being stimulated for the indi- and reprobed with the anti-Akt Ab (lower panel). The positions of the cated times with goat anti-mouse ␬ light chain Abs at a ﬁnal concentration HA-Akt and the faster migrating endogenous Akt are indicated by the of 30 ␮g/ml. B, A20 cells were incubated with 30 nM wortmannin (W), arrows. Note that the “cell lysate” portion of the anti-phospho-Akt blot 0.001% DMSO (D) as a solvent control, or buffer only (Ϫ) for 20 min at (rightmost part, upper panel) was exposed to ﬁlm for a much longer time 37°C before being stimulated with the indicated concentrations of goat than the immunoprecipitation portion of this blot (leftmost part, upper anti-mouse IgG Abs for 10 min. Cell lysates (50 ␮g protein for splenic B cells, 40 ␮g protein for A20 cells) were separated by SDS-PAGE, trans- panel) to visualize the phosphorylation of the HA-Akt. Both parts of the http://www.jimmunol.org/ lower panel were exposed for the same length of time, but were moved ferred to nitrocellulose, and analyzed by immunoblotting with the Ab that apart for the purpose of aligning the lanes. Molecular mass standards (in recognizes Akt that is phosphorylated on serine 473 (upper panel). The kDa) are indicated to the left of each panel. These data represent one of two blots were then stripped and reprobed with the anti-Akt Ab (lower panel). similar experiments. Molecular mass standards (in kDa) are indicated to the left of each panel. Each experiment was repeated twice with similar results.

BCR-induced phosphorylation of Akt is dependent on

PI 3-kinase activity by guest on September 28, 2021 The current model for Akt activation involves recruitment of Akt to the plasma membrane by PIP3 and/or PI(3,4)P2, followed by phosphorylation of Akt at threonine 308 by PDK1 and at serine

473 by PDK2 (26, 70). PIP3 is produced only by PI 3-kinase while PI(3,4)P2 is thought to be produced primarily by the dephosphorylation of PIP3. To test whether BCR-induced phosphorylation of Akt on serine 473 is dependent on activation of PI 3-kinase by the BCR, we asked whether this response could be blocked by the PI 3-kinase inhibitor wortmannin (71). Fig. 3 shows that a 20-min pretreatment with 30 nM wortmannin completely blocked BCR- induced phosphorylation of Akt on serine 473 in both splenic B cells and in the A20 B cell line. Thus, the ability of PDK2 to phosphorylate Akt on this residue in response to BCR engagement is a PI 3-kinase-dependent process.

BCR ligation increases the enzymatic activity of Akt FIGURE 4. BCR ligation activates Akt. A total of 107 RAMOS cells was treated with 30 nM wortmannin (W), 25 ␮M LY294002 (LY), or Although phosphorylation of Akt on serine 473 correlates with buffer only for 20 min at 37°C before being stimulated for the indicated maximal activation of Akt, it was important to directly demon- times with goat anti-human IgM Abs at a ﬁnal concentration of 40 ␮g/ml. strate that BCR engagement increases the enzymatic activity of Cell lysates were immunoprecipitated with an Ab to Akt, and in vitro Akt. To do this, we immunoprecipitated Akt from RAMOS cell kinase assays were performed using Crosstide as a substrate. The incor- lysates and then performed in vitro kinase assays using a substrate poration of 32P into this peptide was determined by spotting the reaction called “Crosstide,” a peptide that contains the sequence from products onto p81 chromatography paper and washing away the free 32 GSK-3 that is phosphorylated by Akt (20). We found that stimu- [␥- P]ATP. Control immunoprecipitations in which the anti-Akt Ab was ϳ lating RAMOS cells for 2–15 min with anti-IgM Abs routinely omitted were performed, and the cpm from these control samples ( 4400 cpm in this experiment) were subtracted from the values for each data caused a 6- to 8-fold increase in Akt enzyme activity (Fig. 4). Akt point. Each data point is the average and range of duplicate samples. Anti- activation was maximal at 2 and 5 min after addition of anti-IgM Akt immunoblots (data not shown) conﬁrmed that equal amounts of Akt Abs and began to decline at 15 min. We also found that BCR- had been precipitated from all of the samples and that no Akt had been induced activation of Akt was dependent on PI 3-kinase activity. precipitated from the control samples in which the anti-Akt Ab had been The PI 3-kinase inhibitors wortmannin and LY294002 both com- omitted from the immunoprecipitation step. These data represent one of pletely blocked the ability of the BCR to activate Akt (Fig. 4). This three similar experiments. The Journal of Immunology 1899

308 was completely blocked by both wortmannin and LY294002 (Fig. 5B), indicating that this response is dependent on PI 3-kinase. Since PDK1 is the only known kinase that can phosphorylate Akt on threonine 308 (31), our results suggest that BCR engagement activates PDK1. Src family tyrosine kinases, as well as the Syk tyrosine kinase, are involved in activation of the PDK2/Akt pathway by the BCR Phosphorylation of Akt on serine 473 is mediated by a kinase that has been termed PDK2, although its identity has not been ﬁrmly established. Using the phosphorylation of Akt on serine 473 as our assay, we investigated some of the upstream requirements for regulation of PDK2 by the BCR. In particular, we focused on the role of the different BCR-associated tyrosine kinases in promoting the phosphorylation of Akt on serine 473. The BCR activates multiple members of the Src family of tyrosine kinases, as well as the Syk and Btk tyrosine kinases (1). Considerable evidence suggests that these three families of ty-

rosine kinases play distinct roles in coupling the BCR to various Downloaded from signaling pathways (72, 73). To investigate the role of these tyrosine kinases in activation of the PDK2/Akt pathway by the BCR, we made use of three variants of the DT40 chicken B cell line, one in which the genes encoding Lyn were disrupted (61), one in which the genes encoding Syk were disrupted (61), and one in which the

FIGURE 5. BCR ligation induces phosphorylation of Akt on threonine genes encoding Btk were disrupted (62). Since Lyn is the only Src http://www.jimmunol.org/ 308. A and B, RAMOS cells were stimulated with 40 ␮g/ml goat anti- family tyrosine kinase expressed in DT40 cells (61), the Lyn-de- human IgM Abs for the indicated times. B, The cells were treated with 30 ficient cells are therefore devoid of all Src kinase activity. nM wortmannin (W), 25 ␮M LY294002 (LY), or buffer only (Ϫ) for 20 Fig. 6A shows that the BCR-induced phosphorylation of Akt on min at 37°C before being stimulated with anti-IgM Abs. C, Small resting serine 473 was completely dependent on the presence of the Src ␮ B cells from mouse spleen were stimulated with 50 g/ml goat anti-IgM family kinase Lyn. In the Lyn-deficient DT40 cells, Akt was not Abs for the indicated times. Cell lysates (60 ␮g protein) were separated by phosphorylated at all on serine 473 in response to BCR engage- SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the Ab that specifically recognizes Akt that is phosphorylated on ment. In contrast, the BCR could induce some phosphorylation of threonine 308 (upper panels). The blots were then stripped and reprobed Akt on serine 473 in the Syk-deficient cells, although this response with the anti-Akt Ab (lower panels). The positions of the phosphorylated was less robust and was not sustained for as long as in the wild- by guest on September 28, 2021 Akt (P-Thr308-Akt) as well as the total Akt population (Akt) are indicated type cells (Fig. 6B). In the wild-type DT40 cells, BCR engagement by arrows. Molecular mass standards (in kDa) are indicated to the left of resulted in strong phosphorylation of Akt that persisted for at least each panel. Each experiment was performed at least twice with similar 30 min (see Fig. 1B). However, in the Syk-deficient DT40 cells, results. BCR engagement consistently resulted in modest phosphorylation of Akt that peaked at 5 min and returned to basal levels by 15 min (Fig. 6B). Thus, in DT40 cells, Lyn is absolutely required for ac- confirms that Akt is a downstream target of PI 3-kinase signaling tivation of the PDK2/Akt pathway by the BCR, while Syk is re- in B cells. quired for maximal phosphorylation of Akt, as well as for sustaining this response. BCR-induced phosphorylation of Akt on threonine 308 The Btk tyrosine kinase is a downstream target of PI 3-kinase in

Previous work has shown that activation of Akt requires that it be that production of PIP3 by PI 3-kinase is required for Btk to trans- phosphorylated on threonine 308 by PDK1 and on serine 473 by locate to the plasma membrane and phosphorylate physiological PDK2. Phosphorylation of threonine 308 may be more important substrates, such as phospholipase C-␥ (7–10, 62). Thus, we con- for Akt activation than phosphorylation of serine 473, since chang- sidered the possibility that Btk might link PI 3-kinase to PDK2, the ing threonine 308 to an alanine completely ablates Akt activation, kinase that phosphorylates Akt on serine 473. Fig. 6C, however, while changing serine 473 to an alanine reduces Akt activation clearly shows that BCR-induced phosphorylation of Akt on serine substantially, but not completely (19, 29, 30). Our finding that 473 is completely normal in Btk-deficient DT40 cells. Thus, Btk is BCR ligation increases the enzymatic activity of Akt suggested not involved in the activation of PDK2 by the BCR. that BCR engagement leads to phosphorylation of Akt on threo- We also showed that DT40 cells lacking PLC-␥2, the only iso- nine 308 as well as on serine 473. At the end of our study, an Ab form of PLC-␥ expressed in these cells (63), exhibited normal Akt that specifically recognizes Akt that is phosphorylated on threo- phosphorylation in response to BCR engagement (Fig. 6D). This nine 308 became available, allowing us to directly test whether supports the idea that BCR-induced phosphorylation of Akt on BCR ligation stimulates phosphorylation of Akt at this site. We serine 473 is dependent solely on the activation of PI 3-kinase and found that BCR engagement resulted in increased phosphorylation does not involve increases in intracellular Ca2ϩ or the activation of of Akt on threonine 308 in both RAMOS cells (Fig. 5A) and small kinases that are regulated by PLC-derived second messengers resting B cells from mouse spleen (Fig. 5C). In the RAMOS cells, (e.g., protein kinase C enzymes). phosphorylation of Akt on threonine 308 was sustained for at least The DT40 experiments described above suggested that Src fam- 60 min after addition of anti-IgM to the cells (Fig. 5A), similar to ily tyrosine kinases, such as Lyn, are both necessary and sufficient what was observed for phosphorylation of serine 473 in these cells for BCR-induced activation of the PDK2/Akt, while Syk is re- (see Fig. 1C). BCR-induced phosphorylation of Akt on threonine quired for sustaining and amplifying this response. To provide 1900 REGULATION OF Akt AND GSK-3 BY THE BCR

FIGURE 7. BCR-induced phosphorylation of Akt in BCR-expressing AtT20 cells involves Fyn and Syk. The 100.33 (BCRϩFynϩ) and Syk13 (BCRϩFynϩSykϩ) variants of the AtT20 endocrine cell line were stimulated with 20 ␮g/ml goat anti-mouse IgM Abs for the indicated times. Aliquots of each cell lysate (15 ␮g protein for anti-phospho-Ser473 Akt blots and anti-Akt blots, 50 ␮g protein for anti-phospho-Thr308 Akt blots) were separated by SDS-PAGE and transferred to nitrocellulose. The ﬁlters were probed with the Ab that recognizes Akt that is phosphorylated on serine 473 (upper panel), the Ab that recognizes Akt that is phosphorylated on threonine 308 (middle panel), or the anti-Akt Ab (lower panel). Mo-

lecular mass standards (in kDa) are indicated to the left of each panel. Downloaded from These data represent one of three similar experiments.

while the Src family tyrosine kinase Fyn was sufﬁcient for BCR- induced Akt phosphorylation in AtT20 cells, Syk was required to

sustain Akt phosphorylation at high levels. These data are consis- http://www.jimmunol.org/ tent with our results using the kinase-deﬁcient DT40 cell variants. In both DT40 cells and AtT20 cells, a Src family tyrosine kinase is sufﬁcient for BCR-induced phosphorylation of Akt on serine 473, while Syk is required for sustaining this response. Thus, Src kinases and Syk are both involved in BCR-induced activation of the PDK2/Akt pathway, but appear to play different roles in this process. We also used the 100.33 and Syk13 variants of the AtT20 cells

to examine the kinase requirements for BCR-induced phosphory- by guest on September 28, 2021 lation of Akt on threonine 308 (Fig. 7). The results obtained were FIGURE 6. BCR-induced phosphorylation of Akt on serine 473 de- very similar to those observed for phosphorylation of Akt on serine pends on Lyn and Syk in DT40 cells. Wild type DT40 cells as well as 473. The Src family tyrosine kinase Fyn was sufficient for BCR- DT40 variants lacking Lyn (A), Syk (B), Btk (C), or PLC-␥2(D) were stimulated with 20 ␮g/ml of the 6E4 anti-chicken light chain mAb (anti-L induced phosphorylation of Akt on threonine 308, while Syk was chain) for the indicated times. Cell lysates (60 ␮g protein) were separated required to sustain this response. Although the Ab that specifically by SDS-PAGE, transferred to nitrocellulose. The filters were probed with recognizes Akt that is phosphorylated on threonine 308 did not the Ab which recognizes Akt that is phosphorylated on serine 473 (upper react with Akt from the DT40 chicken B cell line, the results from panels). The blots were then stripped and reprobed with the anti-Akt Ab the AtT20 cells suggest that regulation of both PDK1 and PDK2 (lower panels). Molecular mass standards (in kDa) are indicated to the left by the BCR involves Syk, as well as a Src family tyrosine kinase. of each panel. Each experiment was repeated twice with similar results. GSK-3␣ and GSK-3␤ are targets of BCR signaling In insulin-responsive cells, GSK-3 is a downstream target of Akt. further evidence to support this conclusion, we made use of several Insulin receptor signaling decreases GSK-3 activity, and this re- variants of the AtT20 endocrine cell line that express a transfected sponse can be blocked by expression of a dominant-negative form murine BCR (␮, ␭, Ig-␣, Ig-␤). Of the tyrosine kinases relevant for of Akt (48). In vitro, both the ␣ and ␤ isoforms of GSK-3 can be BCR signaling, the BCR-expressing AtT20 cells (100.33 cells) phosphorylated by Akt, and this results in a decrease in GSK-3 express only Fyn (67), a Src family tyrosine kinase. We have also enzyme activity (20). Taken together, these data suggest that established a BCR-expressing AtT20 cell line, Syk 13, that ex- GSK-3 may be a physiological substrate of Akt. Since GSK-3 is presses a transfected Syk gene in addition to the endogenous Fyn. involved in a number of important cellular functions, including

Using the 100.33 and Syk13 cells, we asked whether the Src fam- regulation of NF-ATc, our finding that the BCR activated Akt ily tyrosine kinase Fyn was sufficient for BCR-induced activation prompted us to examine the effects of BCR engagement of GSK-3 of the PDK2/Akt pathway and whether Syk influenced this activity and phosphorylation. response. We immunoprecipitated GSK-3␣ using an Ab that specifically In the Fyn-expressing 100.33 cells, we found that BCR engage- recognizes only the ␣ isoform of GSK-3 and then performed in ment led to a transient increase in Akt phosphorylation on serine vitro kinase assays using a peptide derived from glycogen synthase 473 (Fig. 7). Upon addition of anti-IgM Abs to these cells, Akt as a substrate. We found that treating A20 cells with anti-IgG Abs phosphorylation increased slowly, peaking at 15–30 min and then for 2–15 min caused an ϳ50% decrease in GSK-3␣ activity (Fig. declining. In contrast, BCR-induced Akt phosphorylation in the 8A). BCR ligation also decreased GSK-3␣ activity in the RAMOS Syk13 cells (which express both Fyn and Syk) reached maximal human B cell line (Fig. 8B), although to a lesser extent (25–35%) levels after 3 min and remained high for at least 60 min. Thus, than in the A20 cells. The inhibition of GSK-3 activity caused by The Journal of Immunology 1901

FIGURE 8. BCR ligation inhibits GSK-3␣ activity. A, A total of 4 ϫ 106 A20 cells was incubated for 20 min at 37°C with or without 30 nM wortmannin ␮

before being stimulated for the indicated times with goat anti-mouse IgG Abs at a ﬁnal concentration of 40 g/ml. Cell lysates were immunoprecipitated Downloaded from with an Ab to GSK-3␣, and in vitro kinase assays were performed using a peptide derived from glycogen synthase as a substrate. The incorporation of 32P into the peptide was determined by spotting the reaction products onto p81 chromatography paper and washing away the free [␥-32P]ATP. The data are expressed as a percent of the GSK-3␣ activity precipitated from unstimulated cells (8124 Ϯ 20 cpm in this experiment). Each data point is the average and range of duplicate samples. Where no error bars are visible, they were smaller than the symbol. These data represent one of two similar experiments. B, A total of 5 ϫ 106 RAMOS cells were stimulated with goat anti-human IgM Abs for the indicated times. Cell lysates were immunoprecipitated with anti-GSK-3␣ and assayed for GSK-3 activity, as in A. The data from two independent experiments were combined, and each data point represents the mean

and SD for two to four samples (n ϭ no. of samples). http://www.jimmunol.org/

the BCR is similar in magnitude to that caused by insulin (20, 49) that BCR engagement leads to an increase in the levels of PIP3 and

or epidermal growth factor (74), both of which have been shown PI(3,4)P2 in B cells (59). Thus, our data indicate that the BCR to reduce total cellular GSK-3 activity by 40–50%. The ability of activates a PI 3-kinase/Akt/GSK-3 signaling pathway. the BCR to decrease GSK-3␣ activity in the A20 cells was com- Based on our current knowledge (26, 70), the likely series of pletely blocked by the PI 3-kinase inhibitor wortmannin (Fig. 8A). events in this PI 3-kinase/Akt/GSK-3 pathway are: 1) Recruitment ␣ Thus, GSK-3 is a downstream target of PI 3-kinase signaling in of Akt and PDK1 to the plasma membrane via the binding of their by guest on September 28, 2021 B cells. PH domains to PIP and/or PI(3,4)P , 2) activation and/or mem- ␤ 3 2 To determine whether GSK-3 is also a target of BCR signal- brane recruitment of PDK2, 3) phosphorylation of Akt on threo- ing, we made use of a recently developed anti-peptide Ab that nine 308 by PDK1 and on serine 473 by PDK2, which results in ␤ specifically recognizes GSK-3 that is phosphorylated on serine 9. activation of Akt, and 4) release of Akt into the cytoplasm, where This serine residue is phosphorylated by Akt in vitro, and phos- it can phosphorylate GSK-3␣ on serine 21 and GSK-3␤ on serine phorylation of this residue correlates with an inhibition of GSK-3␤ 9, resulting in a decrease in GSK-3 activity. Recently, we have activity (20). Fig. 9 shows that BCR engagement resulted in sub- expressed in WEHI-231 cells an estrogen receptor-Akt chimeric stantial phosphorylation of GSK-3␤. This response was observed protein that can be inducibly activated by addition of 4-hydroxyta- in the A20 murine B cell line, in the RAMOS human B cell line, and in murine splenic B cells. BCR-induced phosphorylation of moxifen to the medium (75). Preliminary experiments have shown GSK-3␤ was inhibited by pretreating the cells with the PI 3-kinase that activation of this estrogen receptor-Akt chimeric protein re- ␣ inhibitor wortmannin (Fig. 9, C and D). Thus, both GSK-3␣ and sults in phosphorylation of GSK-3 , supporting the idea that ␣ GSK-3␤ are PI 3-kinase-dependent targets of BCR signaling. GSK-3 is a downstream target of Akt (B. Hong and M. Gold, unpublished observations). Discussion The use of DT40 chicken B cells and BCR-expressing AtT20 endocrine cells with different subsets of the BCR-regulated ty- In this report, we have shown that the BCR regulates the phos- rosine kinases allowed us to assess the role of Src family kinases, phorylation state and the activity of the Akt and GSK-3 kinases in Syk, and Btk in activation of the PI 3-kinase/PDK2/Akt pathway. a PI 3-kinase-dependent manner. BCR engagement resulted in Together, these studies showed that Src family tyrosine kinases are phosphorylation of Akt on both threonine 308 and serine 473, both necessary and sufficient for activation of this pathway, as modifications that are required for maximal activation of Akt. Moreover, Akt enzyme activity increased 6- to 8-fold after BCR judged by phosphorylation of Akt on serine 473. Akt phosphory- ligation. GSK-3␤ was also phosphorylated in response to BCR lation on serine 473 was completely absent in DT40 cells lacking ligation, in this case on serine 9, a residue that can be phosphor- Lyn, the only Src family tyrosine kinase expressed in these cells. ylated by Akt. Phosphorylation of GSK-3␤ at this site correlates Conversely, both DT40 and AtT20 variants that express a Src fam- with inhibition of its enzymatic activity (20). Finally, we directly ily tyrosine kinase in the absence of Syk were competent for BCR- showed that BCR engagement caused a significant decrease in the induced Akt serine 473 phosphorylation. This latter result indicates activity of the very closely related GSK-3␣ isoform. All of these that activation of Src kinases by the BCR is sufficient for PI 3-ki- responses were blocked by the PI 3-kinase inhibitors wortmannin nase and Akt to be recruited to the plasma membrane and for or LY294002, indicating that both Akt and GSK-3 are downstream PDK2 to be activated and/or localized to the membrane. Several targets of PI 3-kinase-derived lipids. We have previously shown lines of evidence support the conclusion that Src family kinases are 1902 REGULATION OF Akt AND GSK-3 BY THE BCR

signiﬁcant conformational change that reveals the SH3 domain of the kinase (82). Cambier and colleagues (83) have shown that the SH3 domains of Src kinases (including Lyn and Fyn) can bind to proline-rich regions in the p85 subunit of PI 3-kinase, and, in doing so, increase the enzymatic activity of PI 3-kinase. Thus, there are multiple ways in which Src kinases can act independently of Syk to recruit and activate PI 3-kinase. While the Src kinases appear to be essential for initiating the PI 3-kinase/PDK2/Akt pathway, the Syk tyrosine kinase is required for maximal phosphorylation of Akt on serine 473 and for sustaining this response. In the Syk-deﬁcient DT40 cells, the maximal level of Akt phosphorylation of serine 473 was considerably lower than in the wild-type DT40 cells. While this difference in the maximal level of Akt phosphorylation was not as evident in the AtT20 cells, there was clearly a Syk-dependent difference in the kinetics of Akt phosphorylation in both the AtT20 cells and the DT40 cells. In the absence of Syk, Akt phosphorylation was transient, whereas it was sustained at high levels for longer periods of time in the cells

expressing Syk. The precise nature of this requirement for Syk is Downloaded from not clear. One possibility is that both Syk and the Src kinases recruit PI 3-kinase to the plasma membrane, but by different mechanisms. Src kinase-mediated recruitment of PI 3-kinase to the plasma membrane (by the mechanisms described in the previous paragraph) may be transient, while Syk-mediated phosphorylation

of docking proteins, such as Gab1, may be sustained for longer http://www.jimmunol.org/ periods of time. Alternatively, since the readout in these experiments was phosphorylation of Akt on serine 473, PDK2, the kinase that phosphorylates this site, may be subject to regulation by both Src kinases and Syk. Another possibility is that two different kinases can phosphorylate Akt at serine 473, one that is regulated by Src kinases, one that is regulated by Syk. Further characterization of the kinases that phosphorylate Akt on serine 473 is required to address this possibility.

␤ by guest on September 28, 2021 FIGURE 9. BCR-induced phosphorylation of GSK-3 on serine 9. A, Analysis of Akt phosphorylation on threonine 308 in the BCR- ␮ RAMOS cells were stimulated with 40 g/ml goat anti-human IgM Abs; B expressing AtT20 cells showed that the kinase requirements for and C, A20 cells were stimulated with 40 ␮g/ml goat anti-mouse IgG Abs; activation of PDK1 by the BCR were similar to that for PDK2. As and D, Small resting B cells from mouse spleen were stimulated with 30 ␮g/ml goat anti-␬ light chain Abs for the indicated times. C and D, The for phosphorylation of serine 473 by PDK2, Src family tyrosine cells were incubated with (ϩ) or without (Ϫ) 30 nM wortmannin before kinases were sufficient for PDK1-mediated phosphorylation of Akt being stimulated. Cell lysates (40 ␮g) were separated by SDS-PAGE, on threonine 308, while Syk was required for sustained phosphor- transferred to nitrocellulose, and analyzed by immunoblotting with an Ab ylation of threonine 308. Thus, at least in AtT20 cells, PDK1 and that specifically recognizes GSK-3␤ that is phosphorylated on serine 9. To PDK2 appear to be coordinately regulated by both Syk and the Src confirm that equal amounts of GSK-3␤ had been loaded in each lane, the family tyrosine kinase Fyn. The simplest way to explain this co- blots were either reprobed with an anti-GSK-3␤ mAb (A and D)orthe ordinate regulation of PDK1 and PDK2 by Fyn and Syk is that same samples were run on separate gels and analyzed by immunoblotting both of these tyrosine kinases are involved in the membrane re- ␤ with the anti-GSK-3 mAb (B and C). The positions of the phosphorylated cruitment and/or activation of PI 3-kinase by the BCR. However, GSK-3␤ (P-GSK-3␤), as well as the total GSK-3␤ population (GSK-3␤), we cannot rule out the possibility that Fyn or Syk regulate the are indicated by arrows. Note that GSK-3␤ runs as two species on the SDS-PAGE gels. The phosphorylated form of GSK-3␤ appears to comi- subcellular localization and/or activity of PDK1 and PDK2 in grate with the top portion of the upper GSK-3␤ band. Molecular mass other ways. standards (in kDa) are indicated to the left of each panel. In contrast to the role of Src kinases and Syk, we found that the Btk tyrosine kinase was not involved in PDK2-mediated phosphorylation of Akt. BCR-induced phosphorylation of Akt on serine 473 important for the BCR to recruit PI 3-kinase to the plasma mem- was completely normal in Btk-deficient DT40 cells. Moreover, brane, where its substrates are located. First, Lyn has been shown BCR ligation caused significant and sustained phosphorylation of to associate tightly with the cytoplasmic domain of CD19 (76), and Akt in AtT20 cells that express Fyn and Syk but not Btk. BCR-induced activation of Lyn results in tyrosine phosphorylation While we have shown that the BCR regulates the activity of Akt of CD19 and the subsequent binding of PI 3-kinase to CD19 (77). and GSK-3, the role of these enzymes in B cell development and PI 3-kinase may also be recruited to the plasma membrane by activation remain to be elucidated. One of the major functions of using its Src homology (SH) 2 domains to bind to other mem- Akt is to prevent apoptosis. Activation of Akt by PI 3-kinase has brane-associated docking proteins that are phosphorylated on ap- been shown to protect neuronal cells from apoptosis (36, 39). In- propriate tyrosine residues after BCR engagement. Cbl and Gab1 hibition of GSK-3 may also promote cell survival (58). However, are two such docking proteins that bind PI 3-kinase in activated B our observations suggest that BCR-induced activation of Akt and cells (78–80). Tezuka et al. (81) have shown in DT40 cells that the subsequent inhibition of GSK-3 are not sufficient to protect B BCR-induced tyrosine phosphorylation of Cbl is dependent on Lyn cells from apoptosis. BCR ligation causes apoptosis in WEHI-231 but not on Syk. Finally, Src kinase activation is accompanied by a cells, RAMOS cells, and DT40 cells, despite the fact that the BCR The Journal of Immunology 1903 activates Akt and inhibits GSK-3. This implies that the main role Acknowledgments of Akt in BCR signaling is not the prevention of apoptosis. We thank Jason Dinglasan for technical assistance and Tomohiro Kurosaki As opposed to the BCR, it is CD40 that usually delivers B cell for the DT40 cell lines. survival signals. Although CD40 has been reported to activate PI 3-kinase in human B cells (4), our preliminary results show that References CD40 does not induce Akt phosphorylation in resting B cells from 1. Gold, M. R., and L. Matsuuchi. 1995. Signal transduction by the antigen recep- mouse spleen and induces only a very small amount of Akt phos- tors of B and T lymphocytes. Int. Rev. Cytol. 157:181. phorylation (ϳ10% as much as the BCR) in WEHI-231 cells. Ex- 2. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by periments are in progress to test whether CD40 increases Akt en- lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645. 3. DeFranco, A. L. 1997. The complexity of signaling pathways activated by the zyme activity in B cells. Nevertheless, our current results suggest BCR. Curr. Opin. Immunol. 9:296. that activation of Akt does not play a significant role in the ability 4. Aagaard-Tillery, K. M., and D. F. Jelinek. 1996. Phosphatidylinositol 3-kinase of CD40 to prevent B cell apoptosis. Moreover, these results inactivation in normal human B lymphocytes: differential sensitivity of growth and differentiation to wortmannin. J. Immunol. 156:4543. dicate that it is necessary to evaluate whether CD40 activates PI 5. Beckwith, M., R. G. Fenton, I. M. Katona, and D. L. Longo. 1996. Phosphati- 3-kinase in murine B cells. dylinositol-3-kinase activity is required for the anti-Ig-mediated growth inhibition of a human B-lymphoma cell line. Blood 87:202. While BCR-induced Akt activation may not mediate survival 6. Lemmon, M. A., M. Falasca, K. M. Ferguson, and J. Schlessinger. 1997. Regu- signals, a potential role for Akt in B cells may be to enhance latory recruitment of signaling molecules to the cell membrane by pleckstrin- NF-AT -dependent transcription by inhibiting GSK-3. GSK-3 has homology domains. Trends Cell Biol. 7:237. c 7. Bolland, S., R. N. Pearse, T. Kurosaki, and J. V. Ravetch. 1998. SHIP modulates been identified as the kinase that phosphorylates NF-ATc, resulting immune receptor responses by regulating membrane association of Btk. Immunity in its rapid export from the nucleus (51, 52). Inhibition of GSK-3 8:508. Downloaded from may therefore be necessary for NF-AT to accumulate in the nu- 8. Buhl, A. M., C. M. Pleiman, R. C. Rickert, and J. C. Cambier. 1997. Qualitative c regulation of B cell antigen receptor signaling by CD19: selective requirement for cleus and promote transcription. The BCR has been shown to PI3-kinase activation,inositol-1,4,5-trisphosphate production and Ca2ϩ mobiliza- tion. J. Exp. Med. 186:1897. cause NF-ATc to translocate to the nucleus (84, 85), and NF-ATc 2ϩ 9. Scharenberg, A. M., O. El-Hillal, D. Fruman, Z. Li, L. Beitz, S. Lin, I. Gout, is likely to be a key mediator of Ca -dependent signaling initiated L. C. Cantley, D. J. Rawlings, and J. -P. Kinet. 1998. Phosphatidylinositol-3,4,5- by the BCR. Inhibition of GSK-3 also promotes the formation and trisphosphate (PtdIns-3, 4, 5P3)/TEC kinase dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 127:1961. nuclear localization of ␤-catenin/LEF-1 transcription factor com- http://www.jimmunol.org/ 10. Fluckiger, A. -C., Z. Li, R. M. Kato, M. I. Wahl, H. Ochs, R. Longnecker, J. -P. plexes (57). The role of such complexes in BCR signaling has not Kinet, O. N. Witte, A. M. Scharenberg, and D. J. Rawlings. 1998. Btk/Tec ki- been investigated. nases regulate increases in intracellular Ca2ϩ following B cell receptor activation. Another potential role for Akt in BCR signaling is to increase EMBO J. 17:1973. 11. O’Rourke, L. M., R. Tooze, N. Turner, D. M. Sandoval, R. H. Carter, protein synthesis. Akt either directly or indirectly stimulates phos- V. L. J. Tybulewicz, and D. T. Fearon. 1998. CD19 as a membrane-anchored phorylation of 4E-BP1 (44) and the p70S6K kinase (21), both of adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav. Immunity 8:635. which regulate protein translation. 4E-BP1 sequesters the eIF-4E 12. Chong, L. D., A. Traynor-Kaplan, G. M. Bokoch, and M. A. Schwartz. 1994. The translation initiation factor in an inactive complex. Phosphoryla- small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate-5- tion of 4E-BP1 releases eIF-4E and allows it to promote translation kinase in mammalian cells. Cell 79:507. 13. Tolias, K. F., L. C. Cantley, and C. L. Carpenter. 1995. Rho family GTPases bind S6K by guest on September 28, 2021 (46). p70 phosphorylates the ribosomal S6 protein, which re- to phosphoinositide kinases. J. Biol. Chem. 270:17656. sults in increased translation of mRNAs containing 5Ј-terminal 14. Ren, X. D., G. M. Bokoch, A. Traynor-Kaplan, G. H. Jenkins, R. A. Anderson, oligopolypyrimidine tracts (45). Since the mRNAs encoding ribo- and M. A. Schwartz. 1996. Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol-4-phosphate-5-kinase in Swiss 3T3 cells. Mol. Biol. somal proteins contain 5Ј-terminal oligopolypyrimidine tracts, ri- Cell 7:435. bosome formation and general protein translation would also be 15. Nimnual, A. S., B. A. Yatsula, and D. Bar-Sagi. 1998. Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279:560. increased. Finally, Akt may also play a role in regulation of the cell 16. Han, J., K. Luby-Phelps, B. Das, X. Shu, Y. Xia, R. D. Mosteller, U. M. Krishna, cycle by virtue of its ability to increase the activity of the E2F J. R. Falck, M. A. White, and D. Broek. 1998. Role of substrates and products of transcription factor and to promote the transcription and translation PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279:558. of D-type cyclins (43, 47). 17. Franke, T. F., S. -I. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, Our finding that BCR ligation induces phosphorylation of Akt D. R. Kaplan, and P. N. Tsichlis. 1995. The protein kinase encoded by the Akt on both threonine 308 and serine 473 suggests that the BCR acti- proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727. vates both PDK1 and PDK2. Thus, if PDK1 and PDK2 have other 18. Kohn, A. D., K. S. Kovacina, and R. A. Roth. 1995. Insulin stimulates the kinase substrates besides Akt, these proteins are also likely to be targets activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J. 4288:4295. of BCR signaling. Recent work has shown that PDK1 phosphor- 19. Alessi, D. R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and ylates and activates the p70S6K kinase (86), the Sgk protein kinase B. A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin (87), and the ␨ isoform of protein kinase C (PKC) (88). Bras et al. and IGF-1. EMBO J. 15:6541. ␨ 20. Cross, D. A. E., D. R. Alessi, P. Cohen, M. Andjelkovic, and B. A. Hemmings. (89) have shown that BCR ligation causes PKC to translocate 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated protein kinase from the cytosol to the membrane fraction of A20 B lymphoma B. Nature 378:785. cells, suggesting that the BCR activates PKC ␨. However, the roles 21. Burgering, B. M. T., and P. J. Coffer. 1995. Protein kinase B (c-akt) in phosphatidylinositol 3-kinase signal transduction. Nature 376:599. of PI 3-kinase and PDK1 in this process have not been examined. 22. del Peso, L., M. Gonzales-Garcia, C. Page, R. Herrera, and G. Nunez. 1997. The identity of PDK2 has not been firmly established, although Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687. there is some evidence that the ILK can phosphorylate Akt (33). 23. Reif, K., B. M. T. Burgering, and D. A. Cantrell. 1997. Phosphatidylinositol We are now investigating whether the BCR activates ILK. 3-kinase links the interleukin-2 receptor to protein kinase B and p70 S6 kinase. In summary, we have provided evidence that the BCR activates J. Biol. Chem. 272:14426. 24. Scheid, M. P., and V. Duronio. 1998. Dissociation of cytokine-induced phos- a PI 3-kinase/PDK1,2/Akt/GSK-3 signaling pathway, and we have phorylation of Bad and activation of PKB/akt: involvment of MEK upstream of directly shown that BCR engagement regulates the activity of the BAD phosphorylation. Proc. Natl. Acad. Sci. USA 95:7439. Akt and GSK-3 kinases. Since Akt and GSK-3, as well as PDK1 25. Klippel, A., C. Reinhard, W. M. Kavanaugh, G. Apell, M. A. Escobedo, and L. T. Williams. 1996. Membrane localization of phosphatidylinositol 3-kinase is and PDK2, can regulate a number of cellular processes, such as sufficient to activate multiple signal transducing kinase pathways. Mol. Cell. Biol. cell survival, cell cycle progression, transcription, and translation, 16:4117. 26. Coffer, P. J., J. Jin, and J. R. Woodgett. 1998. Protein kinase B (c-Akt): a mul- this signaling pathway is likely to have important roles in BCR tifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem. J. signaling. 335:1. 1904 REGULATION OF Akt AND GSK-3 BY THE BCR

27. Frech, M., M. Andjelkovic, E. Ingley, K. K. Reddy, J. R. Falck, and 55. Welsh, G. I., C. M. Miller, A. J. Loughlin, N. T. Price, and C. G. Proud. 1998. B. A. Hemmings. 1997. High affinity binding of inositol phosphates and phos- Regulation of eukaryotic intiation factor eIF2B: glycogen synthase kinase-3 phoinositides to the pleckstrin homology domain of RAC/Protein kinase B and phosphorylates a conserved serine which undergoes dephosphorylation in re- their influence on the kinase activity. J. Biol. Chem. 272:8474. sponse to insulin. FEBS Lett. 421:125. 28. Andjelkovic, M., D. R. Alessi, R. Meier, A. Fernandez, N. J. C. Lamb, M. Frech, 56. Ikeda, S., S. Kishida, H. Yamamoto, H. Murai, S. Koyama, and A. Kikuchi. 1998. P. Cron, J. M. Lucocq, and B. A. Hemmings. 1997. Role of translocation in the Axin, a negative regulator of the Wnt signaling pathway, forms a complex with activation and function of protein kinase B. J. Biol. Chem. 272:31515. GSK-3␤ and ␤-catenin and promotes GSK-3␤-dependent phosphorylation of 29. Welch, H., A. Eguinoa, L. R. Stephens, and P. T. Hawkins. 1998. PKB and Rac ␤-catenin. EMBO J. 17:1371. are activated in parallel within a PI3-kinase controlled signalling pathway. J. Biol. Chem. 273:11248. 57. Bienz, M. 1998. TCF: transcriptional activator or repressor? Curr. Opin. Cell 30. Bellacosa, A., T. O. Chan, N. N. Ahmed, K. Datta, S. Malstrom, D. Stokoe, Biol. 10:366. F. McCormick, J. Feng, and P. Tsichlis. 1998. Akt activation by growth factors 58. Pap, M., and G. M. Cooper. 1998. Role of glycogen synthase kinase-3 in the is a multi-step process: the role of the PH domain. Oncogene 17:313. phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 31. Alessi, D. R., S. R. James, C. P. Downes, A. B. Holmes, P. R. J. Gaffney, 273:19929. C. B. Reese, and P. Cohen. 1997. Characterization of a 3-phosphoinositide-de- 59. Gold, M. R., and R. Aebersold. 1994. Both phosphatidylinositol 3-kinase and pendent protein kinase which phosphorylates and activates protein kinase B␣. Curr. Biol. 7:261. phosphatidylinositol 4-kinase products are increased by antigen receptor signal- 32. Anderson, K. E., J. Coadwell, L. R. Stephens, and P. T. Hawkins. 1998. Trans- ing in B lymphocytes. J. Immunol. 152:42. location of PDK-1 to the plasma membrane is important in allowing PDK-1 to 60. Paramithiotis, E., K. A. Jacobsen, and M. J. H. Ratcliffe. 1995. Loss of surface activate protein kinase B. Curr. Biol. 8:684. immunoglobulin expression precedes B cell death by apoptosis in the bursa of 33. Delcommenne, M., C. Tan, V. Gray, L. Rue, J. Woodgett, and S. Dedhar. 1998. Fabricius. J. Exp. Med. 181:105. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase ki- 61. Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukuda, H. Yamamura, nase-3 and protein kinase B/Akt by the integrin-linked kinase. Proc. Natl. Acad. and T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor- Sci. USA 95:11211. coupled Ca2ϩ mobiliztion through distinct pathways. EMBO J. 13:1341. 34. Scheid, M. P., R. W. Lauener, and V. Duronio. 1995. Role of phosphatidylinositol 3-OH-kinase activity in the inhibition of apoptosis in hemopoietic cells: 62. Takata, M., and T. Kurosaki. 1995. A role for Bruton’s tyrosine kinase in B cell Downloaded from ␥ phosphatidylinositol 3-OH-kinase inhibitors reveal a difference in signaling be- antigen receptor-mediated activation of phospholipase C- 2. J. Exp. Med. 184: tween interleukin-3 and granulocyte-macrophage colony stimulating factor. Bio- 31. chem. J. 312:159. 63. Takata, M., Y. Homma, and T. Kurosaki. 1995. Requirement of phospholipase 35. Yao, R., and G. M. Cooper. 1995. Requirement for phosphatidylinositol-3 kinase C-␥ 2 activation in surface immunoglobulin M-induced B cell apoptosis. J. Exp. in the prevention of apoptosis by nerve growth factor. Science 267:2003. Med. 182:907. 36. Philpott, K. L., M. J. McCarthy, A. Klippel, and L. L. Rubin. 1997. Activated 64. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of phosphtidylinositol 3-kinase and Akt promote survival of superior cervical neu- high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. rons. J. Cell Biol. 139:809. http://www.jimmunol.org/ USA 90:8393. 37. Franke, T. F., D. R. Kaplan, and L. C. Cantley. 1997. PI3K: downstream AKTion blocks apoptosis. Cell 88:435. 65. Krebs, D. L., Y. Yang, M. Dang, J. Haussmann, and M. R. Gold. 1999. Rapid and 38. Hemmings, B. A. 1997. Akt signaling: linking membrane events to life and death efficient retrovirus-mediated gene transfer into B cell lines. Meth. Cell Sci. In decisions. Science 275:628. press. 39. Dudek, H., S. R. Datta, T. F. Franke, M. J. Birnbaum, R. Yao, G. M. Cooper, 66. Matsuuchi, L., M. R. Gold, A. Travis, R. Grosschedl, A. L. DeFranco, and R. A. Segal, D. R. Kaplan, and M. E. Greenberg. 1997. Regulation of neuronal R. B. Kelly. 1992. The membrane IgM-associated proteins MB-1 and Ig-␤ are survival by the serine-threonine protein kinast Akt. Science 275:661. sufficient to promote surface expression of a partially functional B-cell antigen 40. Datta, S. R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, and receptor in a nonlymphoid cell line. Proc. Natl. Acad. Sci. USA 89:3404. M. E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to 67. Richards, J. D., M. R. Gold, S. L. Hourihane, A. L. DeFranco, and L. Matsuuchi. the cell-intrinsic death machinery. Cell 91:231. 1996. Reconstitution of B cell antigen receptor-induced signaling events in a 41. Cardone, M. H., N. Roy, H. R. Stennicke, G. S. Salvesen, T. F. Franke, E. Stanbridge, S. Frisch, and J. C. Reed. 1998. Regulation of cell death protease nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. J. Biol. by guest on September 28, 2021 caspase-9 by phosphorylation. Science 282:1318. Chem. 271:6458. 42. Brunet, A., A. Bonni, M. J. Zigmond, M. Z. Lin, P. Juo, L. S. Hu, M. J. Anderson, 68. Polakiewicz, R. D., S. M. Schieferl, A. C. Gingras, and M. J. Comb. 1998. K. C. Arden, J. Blenis, and M. E. Greenberg. 1999. Akt promotes cell survival by mu-Opioid receptor activates signaling pathways implicated in cell survival and phosphorylating and inhibiting a forkhead transcription factor. Cell 96:857. translational control. J. Biol. Chem. 273:23534. 43. Brennan, P., J. W. Babbage, B. M. T. Burgering, B. Groner, K. Reif, and 69. Brozinick, J. T., and M. J. Birnbaum. 1998. Insulin, but not contraction, activates D. A. Cantrell. 1997. Phosphatidylinositol 3-kinase couples the interleukin-2 re- Akt/PKB in isolated rat skeletal muscle. J. Biol. Chem. 273:14679. ceptor to the cell cycle regulator E2F. Immunity 7:679. 44. Gingras, A. C., S. G. Kennedy, M. A. O’Leary, N. Sonenberg, and N. Hay. 1998. 70. Downward, J. 1998. Mechanisms and consequences of activation of protein ki- 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by nase B/Akt. Curr. Opin. Cell Biol. 10:262. the Akt (PKB) signaling pathway. Genes Dev. 12:502. 71. Ui, M., T. Okada, K. Hazeki, and O. Hazeki. 1995. Wortmannin as a unique 45. Abraham, R. T. 1998. Mammalian target of rapamycin: immunosuppressive probe for an intracellular signaling protein, phosphoinositide 3-kinase. Trends drugs uncover a novel pathway of cytokine receptor signaling. Curr. Opin. Im- Biochem. Sci. 20:303. munol. 10:330. 72. Kurosaki, T. 1997. Molecular mechanisms in B cell antigen receptor signaling. 46. Pause, A., G. J. Belsham, A. C. Gingras, O. Donze, T. A. Lin, J. C. Lawrence, and Curr. Opin. Immunol. 9:309. N. Sonenberg. 1994. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5Ј-cap function. Nature 371:762. 73. Reth, M., and J. Wienands. 1997. Initiation and processing of signals from the B 47. Muise-Helmericks, R. C., H. L. Grimes, A. Bellacosa, S. E. Malstrom, cell antigen receptor. Annu. Rev. Immunol. 15:453. P. N. Tsichlis, and N. Rosen. 1998. Cyclin D expression is controlled post- 74. Eldar-Finkelman, H., R. Seger, J. R. Vandenheede, and E. G. Krebs. 1995. In- transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. activation of glycogen synthase kinase-3 by epidermal growth factor is mediated J. Biol. Chem. 273:29864. by mitogen-activated protein kinase/p90 ribosomal protein S6 kinase signaling 48. van Weeren, P. C., K. M. T. de Bruyn, A. M. M. de Vries-Smit, J. van Lint, and pathways in NIH/3T3 cells. J. Biol. Chem. 270:987. B. M. T. Burgering. 1998. Essential role for protein kinase B (PKB) in insulin- 75. Kohn, A. D., A. Barthel, K. Kovacina, A. Boge, B. Wallach, S. A. Summers, induced glycogen synthase kinase-3 inactivation. J. Biol. Chem. 273:13150. M. J. Birnbaum, P. H. Scott, J. C. Lawrence, Jr., and R. A. Roth. 1998. Con- 49. Moule, S. K., G. I. Welsh, N. J. Edgell, E. J. Foulstone, C. G. Proud, and struction and characterization of a conditionally active version of the serine/ R. M. Denton. 1997. Regulation of protein kinase B and glycogen synthase ki- threonine kinase Akt. J. Biol. Chem. 273:11937. nase-3 by insulin and ␤-adrenergic agonists in rat epididymal fat cells: activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms. 76. van Noesel, C. J. M., A. C. Lankester, G. M. W. van Schijndel, and J. Biol. Chem. 272:7713. R. A. W. van Lier. 1993. The CR2/CD19 complex on human B cells contains the 50. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT src-family kinase Lyn. Int. Immunol. 5:699. family: regulation and function. Annu. Rev. Immunol. 15:707. 77. Tuveson, D. A., R. H. Carter, S. P. Soltoff, and D. T. Fearon. 1993. CD19 of B 51. Beals, C. R., C. M. Sheridan, C. W. Turck, P. Gardner, and G. R. Crabtree. 1997. cells as a surrogate kinase insert region to bind phosphatidylinositol 3-kinase. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science Science 260:986. 275:1930. 78. Kim, T. J., Y. -T. Kim, and S. Pillai. 1995. Association of activated phosphati- 52. Klemm, J. D., C. R. Beals, and G. R. Crabtree. 1997. Rapid targeting of nuclear dylinositol 3-kinase with p120cbl in antigen receptor-ligated B cells. J. Biol. proteins to the cytoplasm. Curr. Biol. 7:638. Chem. 270:27504. 53. Welsh, G. I., C. Wilson, and C. G. Proud. 1996. GSK3: a SHAGGY frog story. Trends Cell Biol. 6:274. 79. Panchamoorthy, G., T. Fukazawa, S. Miyake, S. Soltoff, K. Reedquist, B. Druker, 54. Utton, M. A., A. Vandecandelaere, U. Wagner, C. H. Reynolds, G. M. Gibb, S. Shoelson, L. Cantley, and H. Band. 1996. p120cbl is a major substrate of C. C. Miller, P. M. Bayley, and B. H. Anderton. 1997. Phosphorylation of tau by tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in glycogen synthase kinase 3␤ affects the ability of tau to promote microtubule vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 assembly. Biochem. J. 323:741. subunit of phosphatidylinositol 3-kinase. J. Biol. Chem. 271:3187. The Journal of Immunology 1905

80. Ingham, R. J., M. Holgado-Madruga, C. Siu, A. J. Wong, and M. R. Gold. 1998. 85. Healy, J. I., R. E. Dolmetsch, L. A. Timmerman, J. G. Cyster, M. L. Thomas, The Gab1 protein is a docking site for multiple proteins involved in signaling by G. R. Crabtree, R. S. Lewis, and C. C. Goodnow. 1997. Different nuclear signals the B cell antigen receptor. J. Biol. Chem. 273:30630. are activated by the B cell receptor during positive versus negative signaling. 81. Tezuka, T., H. Umemori, N. Fusaki, T. Yagi, M. Takata, T. Kurosaki, and Immunity 6:419. T. Yamamoto. 1996. Physical and functional assocation of the cbl protooncogene 86. Alessi, D. R., M. T. Kozlowski, Q. P. Weng, N. Morrice, and J. Avruch,. 1998. product with an Src-family protein tyrosine kinase, p53/p56lyn, in the B cell 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and acti- antigen receptor-mediated signaling. J. Exp. Med. 183:675. vates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 8:69. 82. Moareﬁ, I., M. LaFevre-Bernt, F. Sicheri, M. Huse, C. -H. Lee, J. Kuriyan, and 87. Casamayor, A., P. D. Torrance, T. Kobayashi, J. Thorner, and D. R. Alessi. 1999. W. T. Miller. 1997. Activation of the Src-family tyrosine kinase Hck by SH3 Functional counterparts of mammalian protein kinases PDK1 and DGK in bud- domain displacement. Nature 385:650. ding yeast. Curr. Biol. 9:186. 83. Pleiman, C. M., W. M. Hertz, and J. C. Cambier. 1994. Activation of phospha- 88. Chou, M. M., W. M. Hou, J. Johnson, L. K. Graham, M. H. Lee, C. S. Chen, tidylinositol-3Ј kinase by Src-family kinase SH3 binding to the p85 subunit. A. C. Newton, B. S. Schaffhausen, and A. Toker. 1998. Regulation of protein Science 263:1609. kinase C␨ by PI 3-kinase and PDK-1. Curr. Biol. 8:1069. 84. Venkitaraman, L., D. A. Francis, V. Wang, J. Liu, T. L. Rothstein, and R. Sen. 89. Bras, A., C. Martinez-A., and E. Baixeras. 1997. B cell receptor cross-linking 1994. Cyclosporin A-sensitive induction of NF-AT in murine B cells. Immunity prevents Fas-induced cell death by inactivating the IL-1␤-converting enzyme 1:189. protease and regulating Bcl-2/Bcl-x expression. J. Immunol. 159:3168. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021