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Novel Role of Phosphatidylinositol 3-Kinase in CD28-mediated Costimulation*

Open AccessPublished:March 23, 2001DOI:https://doi.org/10.1074/jbc.M005051200
      Ligation of the CD28 surface receptor provides a major costimulatory signal for full scale T cell activation. Despite extensive studies, the intracellular signaling pathways delivered by CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants were expressed in Jurkat cells. We then measured IL-2 promoter activation after CD28 ligation. Our results showed that the Y189F mutant, which disrupts binding by PI3K, and the YMNM deletion mutant both demonstrated reduced but significant activity for IL-2 promoter activation. In contrast, the N191A mutant, which retains PI3K binding ability, resulted in a complete abrogation of activity, suggesting that PI3K mediates a negative effect upon transcriptional activation of theIL-2 gene. Consistent with this idea, we found that the addition of a PI3K pharmacological inhibitor augmented IL-2 promoter activity, whereas coexpression of a constitutively active form of PI3K reduced this activity. Taken together, these data indicate that PI3K, when associated with the YMNM motif, may act as a negative mediator in CD28-mediated IL-2 gene transcription.
      PI3K
      phosphatidylinositol 3-kinase
      SH2
      Src homology domain 2
      PMA
      phorbol myristate acetate
      IL-2
      interleukin 2
      PAGE
      polyacrylamide gel electrophoresis
      GST
      glutathione S-transferase
      FACS
      fluorescence-activated cell sorting
      In addition to the signaling provided by recognition of antigen-major histocompatibility complex by the T cell receptor, other receptor-ligand interactions play critical roles for full activation of T cells by providing costimulatory signals to T cells. Among them, the CD28-mediated signal is considered to be one of the most important costimulatory signals. Costimulation delivered by CD28 is involved in T cell activation and subsequent expression of T cell functions such as cytokine production (
      • June C.H.
      • Bluestone J.A.
      • Nadler L.M.
      • Thompson C.B.
      ,
      • Sperling A.I.
      • Bluestone J.A.
      ,
      • Boussiotis V.A.
      • Freeman G.J.
      • Gribben J.G.
      • Nadler L.M.
      ). Although the importance of CD28 in T cell activation has been well documented, the intracellular signaling pathways required for CD28-mediated costimulation of T cells have yet to be clearly defined.
      A number of signaling molecules such as phosphatidylinositol 3-kinase (PI3K)1 (
      • Pagés F.
      • Ragueneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ,
      • Prasad K.V.
      • Cai Y.C.
      • Raab M.
      • Duckworth B.
      • Cantley L.
      • Shoelson S.E.
      • Rudd C.E.
      ,
      • Truitt K.E.
      • Hicks C.M.
      • Imboden J.B.
      ,
      • Stein P.H.
      • Fraser J.D.
      • Weiss A.
      ), Grb-2 (
      • Schneider H.
      • Cai Y.C.
      • Prasad K.V.
      • Shoelson S.E.
      • Rudd C.E.
      ), ITK (
      • August A.
      • Gibson S.
      • Kawakami Y.
      • Kawakami T.
      • Mills G.B.
      • Dupont B.
      ), and Tec (
      • Yang W.C.
      • Ghiotto M.
      • Barbarat B.
      • Olive D.
      ) have been shown to be involved in CD28-mediated costimulatory signals. PI3K is a heterodimer, composed of a p85 adaptor subunit linked to a p110 catalytic subunit that phosphorylates the D-3 position of the inositol ring of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, generating phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate, respectively (
      • Wymann M.P.
      • Pirola L.
      ). Ligation of CD28, either by its natural ligands (B7 molecules on antigen-presenting cell) or by monoclonal antibodies, triggers phosphorylation of tyrosine residues in CD28 cytoplasmic domain. Phosphorylation of CD28 on Tyr189within the YMNM motif has been shown to provide a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (
      • Pagés F.
      • Ragueneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ,
      • Prasad K.V.
      • Cai Y.C.
      • Raab M.
      • Duckworth B.
      • Cantley L.
      • Shoelson S.E.
      • Rudd C.E.
      ,
      • Truitt K.E.
      • Hicks C.M.
      • Imboden J.B.
      ,
      • Stein P.H.
      • Fraser J.D.
      • Weiss A.
      ). Through its lipid products, PI3K is involved many cellular responses including proliferation, cell survival, adhesion, and actin rearrangement (
      • Toker A.
      • Cantley L.C.
      ). Since the T cell responses that are induced by CD28-mediated cosignaling overlap the reported functions of PI3K in lymphocyte activation, it is conceivable that PI3K may be the critical signaling molecule in T cell costimulation. For example, wortmannin, a potent inhibitor of PI3K, is reported to inhibit CD28-dependent IL-2 production in human peripheral T cells (
      • Ward S.G.
      • Wilson A.
      • Turner L.
      • Westwick J.
      • Sansom D.M.
      ,
      • Ueda Y.
      • Levine B.L.
      • Huang M.L.
      • Freeman G.J.
      • Nadler L.M.
      • June C.H.
      • Ward S.G.
      ,
      • Ghiotto-Ragueneau M.
      • Battifora M.
      • Truneh A.
      • Waterfield M.D.
      • Olive D.
      ). Furthermore, CD28 mutants, which are unable to bind to PI3K, demonstrate that PI3K is required for CD28-mediated IL-2 production in mouse T cell hybridoma cell lines (
      • Pagés F.
      • Ragueneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ,
      • Cai Y.C.
      • Cefai D.
      • Schneider H.
      • Raab M.
      • Nabavi N.
      • Rudd C.E.
      ). However, conflicting results concerning the requirement for PI3K in CD28-mediated costimulation have been reported. For instance, some investigators reported that wortmannin fails to block CD28-mediated costimulation of IL-2 production by Jurkat cells and murine CD4+ splenic T cells (
      • Ueda Y.
      • Levine B.L.
      • Huang M.L.
      • Freeman G.J.
      • Nadler L.M.
      • June C.H.
      • Ward S.G.
      ,
      • Truitt K.E.
      • Shi J.
      • Gibson S.
      • Segal L.G.
      • Mills G.B.
      • Imboden J.B.
      ,
      • Crooks M.E.
      • Littman D.R.
      • Carter R.H.
      • Fearon D.T.
      • Weiss A.
      • Stein P.H.
      ,
      • Lu Y.
      • Phillips C.A.
      • Trevillyan J.M.
      ,
      • Hutchcroft J.E.
      • Franklin D.P.
      • Tsai B.
      • Harrison-Findik D.
      • Varticovski L.
      • Bierer B.E.
      ). Moreover, they showed that mutation of Tyr189 to Phe, which disrupts PI3K binding, had no effect on the ability of CD28 to deliver a costimulatory signal to Jurkat cells (
      • Truitt K.E.
      • Shi J.
      • Gibson S.
      • Segal L.G.
      • Mills G.B.
      • Imboden J.B.
      ). Therefore, while the role of PI3K in CD28 costimulation has been extensively investigated, there is as yet no consensus about the role of PI3K for CD28 function.
      CD28-mediated costimulation is involved in regulation of various T cell functions such as proliferation, cytokine production, T cell survival, prevention of anergy, and expression of cell surface antigens. We postulate that binding of multiple intracellular signaling molecules to the CD28 receptor may selectively regulate multiple CD28-induced cellular functions. As a part of efforts to define the relationship between CD28-mediated intracellular signaling pathways and T cell functions, we constructed a panel of point and deletion mutants of CD28 including the critical PI3K binding motif, YMNM, and tested their costimulatory ability for activation of IL-2 gene transcription.

      DISCUSSION

      The role of PI3K in CD28 costimulation remains controversial. In this study, we found that the YMNM motif deletion mutant and the Y189F mutant had reduced, but yet retained significant activity for IL-2 promoter activation, whereas the M192L mutant was not altered after CD28 stimulation. Because each of these three mutations eliminated the PI3K association to CD28, we concluded that PI3K is not absolutely required for CD28-mediated IL-2 gene transcription. We also found that the mutation of Asn191 completely abolished CD28-mediated signaling. Since the mutation of Asn191 to Ala reduces Grb-2 binding but does not affect PI3K binding, Grb-2 may have a critical role in CD28-mediated IL-2 gene transcription. Together, these results lead us to propose the following hypothesis. 1) Two molecules that bind to the YMNM motif may control CD28-mediated activation of the IL-2 promoter positively and negatively. 2) Since the N191A mutant retains its ability to bind PI3K and reduces binding to Grb-2, PI3K could be considered to be a negative regulator, while Grb-2 may be a positive regulator, and 3) yet undefined positive regulators may exist, which bind to the CD28 cytoplasmic domain outside of the YMNM motif.
      Several groups demonstrated that PI3K has a crucial role in CD28-mediated IL-2 production. For example, using a similar approach, Cai et al. (
      • Cai Y.C.
      • Cefai D.
      • Schneider H.
      • Raab M.
      • Nabavi N.
      • Rudd C.E.
      ) showed that murine T cell hybridomas expressing point mutations of Tyr189 or Met192within the YMNM motif eliminated IL-2 production after CD28 ligation. In contrast, Truitt et al. (
      • Truitt K.E.
      • Shi J.
      • Gibson S.
      • Segal L.G.
      • Mills G.B.
      • Imboden J.B.
      ) reported that transfection of the mouse CD28 Y189F mutant into Jurkat cells, which showed partial reduction of IL-2-transcriptional activation, did not inhibit CD28-dependent IL-2 production. Furthermore, in Jurkat cells, it was reported that wortmannin treatment did not decrease or, in some cases, even increased CD28-dependent costimulation of IL-2 production (
      • Ueda Y.
      • Levine B.L.
      • Huang M.L.
      • Freeman G.J.
      • Nadler L.M.
      • June C.H.
      • Ward S.G.
      ,
      • Truitt K.E.
      • Shi J.
      • Gibson S.
      • Segal L.G.
      • Mills G.B.
      • Imboden J.B.
      ,
      • Crooks M.E.
      • Littman D.R.
      • Carter R.H.
      • Fearon D.T.
      • Weiss A.
      • Stein P.H.
      ,
      • Lu Y.
      • Phillips C.A.
      • Trevillyan J.M.
      ,
      • Hutchcroft J.E.
      • Franklin D.P.
      • Tsai B.
      • Harrison-Findik D.
      • Varticovski L.
      • Bierer B.E.
      ). Truitt et al. (
      • Truitt K.E.
      • Shi J.
      • Gibson S.
      • Segal L.G.
      • Mills G.B.
      • Imboden J.B.
      ) also found that wortmannin did not inhibit CD28-dependent IL-2 production from freshly isolated murine CD4+ T cells. On the contrary, wortmannin was reported to inhibit CD28-mediated costimulation of IL-2 in human primary T cells (
      • Ward S.G.
      • Wilson A.
      • Turner L.
      • Westwick J.
      • Sansom D.M.
      ,
      • Ueda Y.
      • Levine B.L.
      • Huang M.L.
      • Freeman G.J.
      • Nadler L.M.
      • June C.H.
      • Ward S.G.
      ,
      • Ghiotto-Ragueneau M.
      • Battifora M.
      • Truneh A.
      • Waterfield M.D.
      • Olive D.
      ). One of the possible explanations for the apparent discrepancies among these reports may be the nature of the cell used in the respective studies. We have recently found that T cells from transgenic mice expressing the CD28 Y189F mutation are able to produce a significant amount of IL-2 following CD28 cross-linking.
      Harada, Y., Tokushima, M., Matsumoto, Y., Ogawa, S., Otsuka, M., Hayashi, K., Weiss, B. D., June, C. H., and Abe, R. (2001) J. Immunol., in press.
      This result suggests that PI3K is dispensable for CD28-mediated IL-2 production in primary T cells.
      Recently, two groups generated PI3K p85α null mutant mice and reported that these mice had impaired B cell development and functions, whereas T cell development and proliferation were normal (
      • Suzuki H.
      • Terauchi Y.
      • Fujiwara M.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      • Koyasu S.
      ,
      • Fruman D.A.
      • Snapper S.B.
      • Yballe C.M.
      • Davidson L., Yu, J.Y.
      • Alt F.W.
      • Cantley L.C.
      ). These reports are consistent with our conclusion that PI3K (p85α)-CD28 association is not obligatory for CD28-mediated T cell activation.
      The physiological significance of the inhibition of IL-2 promoter activity by PI3K is unclear at present. The serine/threonine kinase PKB/Akt is a downstream effector for PI3K. Recently, the PI3K-Akt pathway was shown to inhibit the Raf-Mek-ERK pathway depending on the differentiation stage of muscle cell (
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ). Akt activation inhibited the Raf-Mek-ERK pathway in differentiated myotubes, but not in their myoblast precursors. In Jurkat cells, PI3K may inhibit IL-2 promoter activation using the same pathway. We are presently studying whether this inhibitory pathway exists in Jurkat cells. Given that the PI3K-Akt pathway inhibits the Raf-Mek-ERK pathway depending on the differentiation stage of muscle cells, it is possible that PI3K might play a differential role depending on the activation state of the T cell. In fact, distinct functions of CD28-mediated signals during T cell antigen recognition and activation have been documented (
      • Lenschow D.J.
      • Walunas T.L.
      • Bluestone J.A.
      ). Our recent study using CD28 null mice that have been reconstituted with CD28 mutants supports this hypothesis. Namely, splenic T cells expressing the CD28 Y189F mutant are deeply impaired in the proliferative response and IL-2 production induced by CD28-ligation and CD3 stimulation at 24 h after stimulation, whereas these responses dramatically improved at 48 h after stimulation.2 From these results, it is conceivable that PI3K activation by CD28 engagement may suppress IL-2 production in late but not in early activation stages and therefore, may lead T cell responses to terminate. In this scenario, the phenomenon observed in the present study using Jurkat cells may reflect IL-2 production in the late activation state of T cells.
      CTLA-4, a member of the CD28 family of receptors, appears to send a negative signal to T cells (
      • Walunas T.L.
      • Lenschow D.J.
      • Bakker C.Y.
      • Linsley P.S.
      • Freeman G.J.
      • Green J.M.
      • Thompson C.B.
      • Bluestone J.A.
      ,
      • Krummel M.F.
      • Allison J.P.
      ,
      • Kearney E.R.
      • Walunas T.L.
      • Karr R.W.
      • Morton P.A.
      • Loh D.Y.
      • Bluestone J.A.
      • Jenkins M.K.
      ,
      • Krummel M.F.
      • Sullivan T.J.
      • Allison J.P.
      ). CTLA-4 also has a YXXM motif in the cytoplasmic domain and Schneider et al. (
      • Schneider H.
      • Prasad K.V.
      • Shoelson S.E.
      • Rudd C.E.
      ) reported that anti-CTLA-4 antibody ligation resulted in detectable levels of PI3K activity in human T lymphoblastoid cell line and in PHA-stimulated peripheral T cells. This result suggests the possibility that CTLA-4-induced suppression of IL-2 production may be mediated by the negative function of PI3K. Recently, a third CD28 family receptor, ICOS, has been identified (
      • Hutloff A.
      • Dittrich A.M.
      • Beier K.C.
      • Eljaschewitsch B.
      • Kraft R.
      • Anag-nostopoulos I.
      • Kroczek R.A.
      ,
      • Yoshinaga S.K.
      • Whoriskey J.S.
      • Khare S.D.
      • Sarmiento U.
      • Guo J.
      • Horan T.
      • Shih G.
      • Zhang M.
      • Coccia M.A.
      • Kohno T.
      • Tafuri-Bladt A.
      • Brankow D.
      • Campbell P.
      • Chang D.
      • Chiu L.
      • Dai T.
      • Duncan G.
      • Elliott G.S.
      • Hui A.
      • McCabe S.M.
      • Scully S.
      • Shahinian A.
      • Shaklee C.L.
      • Van G.
      • Mak T.W.
      • et al.
      ,
      • Tamatani T.
      • Tezuka K.
      • Hanzawa-Higuchi N.
      ). This molecule also possesses a PI3K binding motif, YMFM. We have generated and expressed chimeric molecules composed of the CD28 extracellular domain and ICOS intracellular domain, and found that cross-linking of these molecules by anti-murine CD28 antibody resulted in a specific association with PI3K, whereas only slight IL-2 promoter activity was detected. This result is consistent with the notion of a non-obligatory role of PI3K for IL-2 production.
      The costimulatory effects of CD28 are not limited to IL-2 production, and PI3K is also involved in many cellular responses. Thus, PI3K may have a critical role in T cell responses augmented by CD28 costimulation other than IL-2 production. For example, it has been shown that CD28 costimulation enhances T cell survival following T cell receptor stimulation by increasing the expression of Bcl-XL(
      • Boise L.H.
      • Minn A.J.
      • Noel P.J.
      • June C.H.
      • Accavitti M.A.
      • Lindsten T.
      • Thompson C.B.
      ). PI3K was also shown to be involved in cellular survival through activation of Akt. Akt is known to block apoptosis through phosphorylation and inactivation of the Bcl-2 family protein Bad, caspase-9, and Forkhead family members (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). By the activation of PI3K, CD28 may block apoptosis through the PI3K-Akt pathway. Furthermore, CD28 costimulation can also affect adhesion between T cells and antigen-presenting cells, which is important for efficient T cell antigen recognition. Indeed, PI3K has been implicated in CD28-dependent increases in β1-integrin-mediated adhesion to fibronectin (
      • Zell T.
      • Hunt S.W.R.
      • Mobley J.L.
      • Finkelstein L.D.
      • Shimizu Y.
      ). In summary, it is likely that the binding of multiple signaling molecules to the CD28 cytosolic domain may selectively regulate diverse cellular functions.

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