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 phosphor- ylation 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-g (PLC-g), 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 do- main 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-g-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 g 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-g (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-g (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-g, phospholipase naling proteins (6). Thus, it is likely that the activation of PI 3-kinase g 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 syn- thase kinase-3; HA, hemagglutinin. other signaling pathways in addition to the PLC-g pathway. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1895 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-3a on serine 21 and GSK-3b 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-3a and GSK-3b may be physiological substrates domain that binds the PI 3-kinase-derived lipids PIP and PI 3,4- 3 of Akt.