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Co-ligation of the Antigen and Fc Receptors Gives Rise to the Selective Modulation of Intracellular Signaling in B Cells

REGULATION OF THE ASSOCIATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND INOSITOL 5′-PHOSPHATASE WITH THE ANTIGEN RECEPTOR COMPLEX*
Open AccessPublished:February 07, 1997DOI:https://doi.org/10.1074/jbc.272.6.3838
      Cross-linking of the Fc receptor (FcR) to surface immunoglobulin (sIg) on B cells inhibits the influx of extracellular calcium and abrogates the proliferative signal. The mechanism by which this occurs is not well understood. In this report we show that co-cross-linking the FcR to the antigen receptor gives rise to very selective modulation of signal transduction in B cells. Co-cross-linking sIg and the FcR enhanced the phosphorylation of the FcR, the adapter protein, Shc, and the inositol 5′-phosphatase Ship. Furthermore, phosphorylation of the FcR induced its association with Ship. Cross-linking of the FcR and sIg decreased the tyrosine phosphorylation of CD19, which led to a reduction in the association of phosphatidylinositol 3-kinase. In addition, the phosphorylation of several other proteins of 73, 39, and 34 kDa was reduced. Activation of the cells with either F(ab′)2 or intact anti-IgG induced very similar changes in levels of tyrosine phosphorylation of most other proteins, and no differences in the activation of several protein kinases were observed. These results indicate that the inhibitory signal that is transmitted through the FcR is not mediated by a global shutdown of tyrosine phosphorylation but is, rather, a selective mechanism involving localized changes in the interactions of adapter proteins and the enzymes Ship and phosphatidylinositol 3-kinase with the antigen receptor complex.

      INTRODUCTION

      Activation of B cells through the antigen receptor gives rise to a sequence of intracellular signals that leads to the proliferation and/or differentiation of the cells (reviewed in Refs.
      • Gold M.R.
      • DeFranco A.L.
      and
      • Cambier J.C.
      • Pleiman C.M.
      • Clark M.R.
      ). In contrast, concomitant activation of B cells through both the sIg
      The abbreviations used are: sIg
      surface immunoglobulin
      FcR
      Fc receptor
      CHAPS
      3-[(3-chloramidopropyl)dimethylammonio]-1-propane-sulfonate
      PBS
      phosphate-buffered saline
      PAGE
      polyacrylamide gel electrophoresis
      IL
      interleukin
      PLC
      phospholipase C
      PI3
      phosphatidylinositol 3
      JAK
      Janus kinase
      SHP
      Src homology 2-containing phosphatase.
      and Fc receptors leads to a dominant negative signal that inhibits activation of the cells (
      • Sidman C.L.
      • Unanue E.R.
      ,
      • Klaus G.G.
      • Hawrylowicz C.M.
      • Holman M.
      • Keeler K.D.
      ,
      • Phillips N.E.
      • Parker D.C.
      ,
      • Phillips N.E.
      • Parker D.C.
      ). Thus the role of the FcR on B lymphocytes is to provide a powerful mechanism for the cells to distinguish between free antigen and antigen-antibody immune complexes.
      The inhibitory pathway triggered following co-ligation of the sIg and FcR is not well understood. Studies have recently been undertaken to try to distinguish the molecular events that give rise to the differences in signaling mediated through the antigen receptor alone from that following the cross-linking of sIg to the FcR. Stimulation of the cells with F(ab′)2 anti-IgG, which mimics antigen binding to the receptor, gives rise to the mobilization of calcium within the cell from both intracellular and extracellular pools. Uptake of extracellular calcium appears to be critical for the outcome of activation. Co-cross-linking of the FcR to sIg with intact anti-Ig, which mimics the binding of antibody-antigen complexes, inhibits the influx of extracellular calcium (
      • Choquet D.
      • Partiseti M.
      • Amigorena S.
      • Bonnerot C.
      • Fridman W.H.
      • Korn H.
      ,
      • Diegel M.L.
      • Rankin B.M.
      • Bolen J.B.
      • Dubois P.M.
      • Kiener P.A.
      ). Additionally, co-ligation of the sIg and Fc receptors subsequent to the opening of the plasma membrane calcium channel stimulates the closure of the channel (
      • Diegel M.L.
      • Rankin B.M.
      • Bolen J.B.
      • Dubois P.M.
      • Kiener P.A.
      ), and the level of intracellular calcium rapidly declines. In A20 cells, it is possible to restore the influx of extracellular calcium and to overcome the FcR-mediated inhibition of cell activation and IL-2 production by low concentrations of calcium ionophore (
      • Diegel M.L.
      • Rankin B.M.
      • Bolen J.B.
      • Dubois P.M.
      • Kiener P.A.
      ).
      Activation of B cells through the antigen receptor also results in the tyrosine phosphorylation of many proteins, including the cell surface antigens CD19 and CD22 that are associated with the antigen receptor (
      • Chalupny N.J.
      • Kanner S.B.
      • Schieven G.L.
      • Wee S.F.
      • Gilliland L.K.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Roifman C.M.
      • Ke S.
      ,
      • Bhakdi S.
      • Tranum-Jensen J.
      ,
      • Schulte R.J.
      • Campbell M.-A.
      • Fischer W.H.
      • Sefton B.M.
      ), the components of the antigen receptor complex, Igα and Igβ (
      • Sanchez M.
      • Misulovin Z.
      • Burkhardt A.L.
      • Mahajan S.
      • Costa T.
      • Franke R.
      • Bolen J.B.
      • Nussenzweig M.
      ,
      • Clark M.R.
      • Johnson S.A.
      • Cambier J.C.
      ,
      • Clark M.R.
      • Campbell K.S.
      • Kazlauskas A.
      • Johnson S.A.
      • Hertz M.
      • Potter T.A.
      • Pleiman C.
      • Cambier J.C.
      ), the tyrosine kinases Syk, Lyn, and Btk (
      • Bolen J.B.
      ,
      • Yamanashi Y.
      • Kakiuchi T.
      • Mizuguchi J.
      • Yamamoto T.
      • Toyoshima K.
      ,
      • Rowley R.B.
      • Burkhardt A.L.
      • Chao H.-G.
      • Matsueda G.R.
      • Bolen J.B.
      ), the phospholipases PLCγ1 and PLCγ2 (
      • Carter R.H.
      • Park D.J.
      • Rhee S.G.
      • Fearon D.T.
      ,
      • Hempel W.M.
      • Schatzman R.C.
      • DeFranco A.L.
      ,
      • Coggeshall K.M.
      • McHugh J.C.
      • Altman A.
      ), and the adapter and signaling proteins Cbl, Vav, Shc, and Gap (
      • Sidman C.L.
      • Unanue E.R.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.
      • Band H.
      ,
      • Tezuka T.
      • Umemori H.
      • Fusaki N.
      • Yagi T.
      • Takata M.
      • Kurosaki T.
      • Yamamoto T.
      ,
      • Cory G.O.C.
      • Lovering R.C.
      • Hinshelwood S.
      • MacCarthy-Morrogh L.
      • Levinsky R.J.
      • Kinnon C.
      ,
      • Lankester A.C.
      • van Schijndel G.M.
      • Rood P.M.
      • Verhoeven A.J.
      • Van Lier R.A.
      ,
      • Smit L.
      • van der Horst G.
      • Borst J.
      ,
      • Nagai K.
      • Takata M.
      • Yamamura H.
      • Kurosaki T.
      ,
      • Weng W.K.
      • Jarvis L.
      • LeBien T.W.
      ,
      • Gold M.R.
      • Crowley M.T.
      • Martin G.A.
      • McCormick F.
      • DeFranco A.L.
      ). Phosphorylation and activation of PLCγ1 and PLCγ2 results in the generation of inositol 1,4,5-trisphosphate, which in turn stimulates the release of calcium from intracellular stores. However, the signaling pathway that regulates the opening and closing of the plasma membrane calcium channel has not been well established.
      Co-cross-linking of the FcR to sIg also gives rise to the phosphorylation of many intracellular proteins, including the FcR itself, on a tyrosine residue in the intracellular domain (
      • Muta T.
      • Kurosaki T.
      • Misulovin Z.
      • Sanchez M.
      • Nussenzweig M.C.
      • Ravetch J.V.
      ). The region containing this tyrosine residue is essential for the inhibitory activity of the FcR (
      • Muta T.
      • Kurosaki T.
      • Misulovin Z.
      • Sanchez M.
      • Nussenzweig M.C.
      • Ravetch J.V.
      ). Subsequently, it has been found that phosphorylation of the FcR on this tyrosine residue results in the interaction of the receptor with the Src homology 2-containing tyrosine phosphatase SHP-1 (
      • D'Ambrosio D.
      • Hippen K.L.
      • Minskoff S.A.
      • Mellman I.
      • Pani G.
      • Siminovitch K.A.
      • Cambier J.C.
      ,
      • Pani G.
      • Kozlowski M.
      • Cambier J.C.
      • Mills G.B.
      • Siminovitch K.A.
      ). This association, which may give rise to activation of the phosphatase, is then thought to provide the signal that shuts off activation of the receptor complex and thus to abrogate the proliferation of the cells.
      SHP-1 has been found to be associated with the antigen receptor in unstimulated cells (
      • Pani G.
      • Kozlowski M.
      • Cambier J.C.
      • Mills G.B.
      • Siminovitch K.A.
      ,
      • Cyster J.G.
      • Goodnow C.C.
      ) and with CD22 in cells activated through sIg (
      • Lankester A.C.
      • van Schijndel G.M.W.
      • van Lier R.A.W.
      ). Cross-linking of CD22 with antibodies can modulate signaling through the antigen receptor (
      • Doody G.M.
      • Justement L.B.
      • Delibrias C.C.
      • Matthews R.J.
      • Lin J.
      • Thomas M.L.
      • Fearon D.T.
      ), and it has been proposed that one mechanism for this is that ligation of CD22, by antibodies or counter receptors on adjacent cells, gives rise to the removal of the SHP-1 from the immediate locale of the sIg signaling complex, thus removing the inhibitory enzyme (
      • Doody G.M.
      • Justement L.B.
      • Delibrias C.C.
      • Matthews R.J.
      • Lin J.
      • Thomas M.L.
      • Fearon D.T.
      ,
      • Clark E.A.
      ). Thus phosphorylation of CD22 and the FcR and their interactions with SHP-1 may provide opposing regulatory control on the strength or duration of the signal transmitted through the antigen receptor (
      • Cyster J.G.
      • Goodnow C.C.
      ,
      • Doody G.M.
      • Justement L.B.
      • Delibrias C.C.
      • Matthews R.J.
      • Lin J.
      • Thomas M.L.
      • Fearon D.T.
      ,
      • Clark E.A.
      ).
      The events that follow the phosphorylation of the FcR and its association with SHP-1 have not been well characterized. The phosphorylated proteins that are the targets of the phosphatase in this regulatory pathway have not been identified, and the influence of the SHP-1 and FcR interaction on the activation of downstream signal transduction pathways has not been determined. Additionally, it is not known how the phosphorylation of the FcR and its subsequent interaction with SHP-1 leads to the rapid closure of the calcium channel.
      In this report we have compared the signaling events that occur following triggering of the antigen receptor either alone or with co-ligation with the FcR. We have found that the phosphorylation and activation of many proteins in the signaling pathways were not significantly different between the two modes of stimulation; however, there were marked changes in the phosphorylation states of a few proteins, including CD19, FcR, Ship, and Shc. These changes resulted in the association of the Ship with the FcR complex and a loss of association of PI3-kinase with CD19.
      These results indicate that the inhibitory signal mediated by the FcR on B cells is a very selective modulation of intracellular signaling and is not simply a consequence of the global dephosphorylation of the signaling components of the antigen receptor by the association of SHP-1 with the receptor complex. Rather, the co-ligation of sIg and the FcR gives rise to changes in the phosphorylation state of a limited set of proteins, which then alters their ability to interact with components of the antigen receptor. The results also suggest that the local alterations in phosphatidylinositol phosphate metabolism by enzymes associated with the antigen receptor complex, namely PI3-kinase and Ship, may be critical in the regulation of the opening and closure of the plasma membrane calcium channel.
      While this manuscript was in preparation, Chacko and co-workers (
      • Chacko G.W.
      • Tridandapani S.
      • Damen J.E.
      • Liu L.
      • Krystal G.
      • Coggeshall K.M.
      ) and Ono and co-workers (
      • Ono M.
      • Bolland S.
      • Tempst P.
      • Ravetch J.V.
      ) also reported finding the enhanced phosphorylation of Ship and its association with Shc and the FcR following co-ligation of sIg and the FcR.

      DISCUSSION

      The FcR plays a crucial role in the regulation of activation of B cells (
      • Sidman C.L.
      • Unanue E.R.
      ,
      • Klaus G.G.
      • Hawrylowicz C.M.
      • Holman M.
      • Keeler K.D.
      ,
      • Phillips N.E.
      • Parker D.C.
      ,
      • Phillips N.E.
      • Parker D.C.
      ). Co-cross-linking the antigen and Fc receptors gives rise to the phosphorylation of the receptor and the generation of a dominant inhibitory signal that abrogates the proliferation or differentiation of the lymphocytes (
      • Muta T.
      • Kurosaki T.
      • Misulovin Z.
      • Sanchez M.
      • Nussenzweig M.C.
      • Ravetch J.V.
      ). FcR phosphorylation promotes its association with other intracellular proteins such as SHP-1 (
      • D'Ambrosio D.
      • Hippen K.L.
      • Minskoff S.A.
      • Mellman I.
      • Pani G.
      • Siminovitch K.A.
      • Cambier J.C.
      ,
      • Pani G.
      • Kozlowski M.
      • Cambier J.C.
      • Mills G.B.
      • Siminovitch K.A.
      ,
      • Cyster J.G.
      • Goodnow C.C.
      ) via Src homology 2 interactions. It has been proposed that the association between SHP-1 and the FcR activates the phosphatase, which then shuts down the signaling through the antigen receptor (
      • D'Ambrosio D.
      • Hippen K.L.
      • Minskoff S.A.
      • Mellman I.
      • Pani G.
      • Siminovitch K.A.
      • Cambier J.C.
      ,
      • Pani G.
      • Kozlowski M.
      • Cambier J.C.
      • Mills G.B.
      • Siminovitch K.A.
      ). It has also been shown that co-cross-linking sIg to the FcR prevents the influx of extracellular calcium by closing the plasma membrane calcium channel (
      • Diegel M.L.
      • Rankin B.M.
      • Bolen J.B.
      • Dubois P.M.
      • Kiener P.A.
      ). However, it is not clear how co-cross-linking the FcR to sIg abrogates the influx of calcium and whether the association of activated SHP-1 with the receptor complex curtails all of the activation signals mediated by the antigen receptor or if the effect is more restricted.
      In this study we show that co-cross-linking sIg to the FcR gives rise to very selective modulation of the signaling pathways stimulated by the antigen receptor. Co-ligation of sIg and the FcR neither inhibits nor reverses the stimulation of phosphorylation of many of the intermediate proteins thought to be involved in signaling through the antigen receptor. There was no detectable difference in the phosphorylation of Cbl, Vav, Fyn, Gap, Syk, PI3-kinase Igβ, and CD22 between activation of the cells through either receptor. Phosphorylation of the Igα chain of the antigen receptor complex was also induced by triggering either with F(ab′)2 or intact anti-IgG, although the levels of phosphorylation stimulated by the latter treatment were slightly diminished. The significance of this is unclear at present.
      It has been shown that both PLCγ1 and PLCγ2 become phosphorylated and activated following stimulation of the cells through the antigen receptor (
      • Carter R.H.
      • Park D.J.
      • Rhee S.G.
      • Fearon D.T.
      ,
      • Hempel W.M.
      • Schatzman R.C.
      • DeFranco A.L.
      ,
      • Coggeshall K.M.
      • McHugh J.C.
      • Altman A.
      ). This then generates the formation of inositol trisphosphate, which then leads to the release of calcium from intracellular stores. In addition, co-cross-linking sIg to the FcR curtails the influx of extracellular calcium but does not significantly inhibit the mobilization of calcium from intracellular stores (
      • Choquet D.
      • Partiseti M.
      • Amigorena S.
      • Bonnerot C.
      • Fridman W.H.
      • Korn H.
      ,
      • Diegel M.L.
      • Rankin B.M.
      • Bolen J.B.
      • Dubois P.M.
      • Kiener P.A.
      ). In agreement with these observations, our experiments showed that co-cross-linking sIg to the FcR had no significant effect on the phosphorylation of PLCγ1. Phosphorylation of PLCγ2 was still significantly enhanced over unstimulated cells, although the levels were slightly decreased. These results are in contrast to a recent report using murine splenic B cells, in which activation of PLCγ2 was significantly inhibited upon co-ligation of the FcR to sIg (
      • Sarkar S.
      • Schlottmann K.
      • Cooney D.
      • Coggeshall K.M.
      ). The reason for this disparity is not clear. It may reflect the differences in cell populations that were used or the conditions of activation. However, it is difficult to directly compare the two studies, since in the other report the influence of the signals on calcium mobilization from the intracellular and extracellular stores was not described.
      Stimulation of B cells through sIg gives rise to the rapid ordered activation of a cascade of protein kinases (
      • Bolen J.B.
      ,
      • Yamanashi Y.
      • Kakiuchi T.
      • Mizuguchi J.
      • Yamamoto T.
      • Toyoshima K.
      ,
      • Rowley R.B.
      • Burkhardt A.L.
      • Chao H.-G.
      • Matsueda G.R.
      • Bolen J.B.
      ,
      • Lee J.C.
      • Young P.R.
      ,
      • Nick J.A.
      • Avdi N.J.
      • Gerwins P.
      • Johnson G.L.
      • Worthen G.S.
      ,
      • Saouaf S.J.
      • Mahajan S.
      • Rowley R.B.
      • Kut S.A.
      • Fargnoli J.
      • Burkhardt A.L.
      • Tsukada S.
      • Witte O.N.
      • Bolen J.B.
      ,
      • Weiss A.
      • Littman D.R.
      ). No differences were observed in the activation of the kinases Syk, Lyn, Fyn, Btk, Erk1, Erk2, and c-Jun amino-terminal kinase 1, between stimulation of the cells through either sIg alone or co-ligation of the FcR to sIg (data not shown). This suggests that these enzymes are not the target for FcR-associated SHP-1.
      Co-cross-linking Fc and antigen receptors did give rise to some very specific changes in the signal transduction pathway. As reported previously (
      • Muta T.
      • Kurosaki T.
      • Misulovin Z.
      • Sanchez M.
      • Nussenzweig M.C.
      • Ravetch J.V.
      ), stimulation of the lymphocytes with intact anti-Ig gave rise to a marked increase in the phosphorylation of the FcR. Immunoprecipitation of the FcR from activated cells also revealed other changes in the cells. A decrease in the phosphorylation of three proteins that co-precipitated with the immune complexes was consistently observed. These proteins, of about 34, 39, and 73 kDa, could also be detected in other immunoprecipitates (see Figs. 2A and 4A). The identity of these proteins is not known, but preliminary experiments have indicated that they are not the Igα and Igβ chains, Syk, SHP-1, SHP-2, or Raf-1. In addition to these changes, it was also possible to detect an increase in the level of a phosphoprotein of about 150 kDa in the immunoprecipitates of the FcR from cells activated with intact anti-IgG.
      Immunoprecipitation of Shc or Grb2 following activation of the B cells by co-ligation of sIgG and FcR revealed that there was enhanced phosphorylation of a doublet centered around 52 kDa, corresponding to Shc. In addition, immunoprecipitates of both Grb2 and Shc from cells activated by co-cross-linking sIg and FcR showed an increase in the levels of an associated phosphoprotein of about 150 kDa. Analysis of the immunoprecipitates of Shc, Grb2, and FcR indicated that this protein was Ship, the inositol 5′-phosphatase (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ,
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ). Subsequently, immunoprecipitation of Ship revealed that the phosphorylation of the enzyme increased following activation of cells through sIg alone; however, the level of phosphorylation was further significantly enhanced when the cells were activated by co-ligation of sIg and FcR. Ship can associate with Shc through the phosphotyrosine binding domain of the latter (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ,
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ), and there are also several proline-rich sequences within Ship that may allow interaction with the SH3 domains of other proteins such as Grb2 (
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ). Since Shc interacts with Grb2, Ship could also indirectly associate with Grb2 via a mutual interaction with Shc. Stimulation of an increase in the phosphorylation of Ship could give rise to an enhanced association with Shc and thereby Grb2. These interactions would account for the increased level of Ship seen in immunoprecipitates of Shc and Grb2 in this study. Alternatively the phosphorylation of Ship may promote the interaction of the enzyme with the Src homology 2 domains of Grb2 or Shc. At present the factors regulating these interactions have not been elucidated.
      Ship was also found present in the immunoprecipitates of the FcR after stimulation of the cells by co-ligation of antigen and Fc receptors. It could not be detected in immunoprecipitates of FcR from cells activated through sIgG alone. Additionally, immunoprecipitates of Ship from cells stimulated with intact anti-IgG revealed the presence of a phosphoprotein that closely resembled the FcR. These results suggest that the phosphorylated FcR can associate with Ship, probably through interaction with the Src homology 2 domain of the enzyme. A biotinylated peptide corresponding to the sequence surrounding the phosphorylated tyrosine in the cytoplasmic domain precipitated Ship from the lysate of unstimulated cells, whereas the nonphosphorylated form of the peptide did not. This shows that the phosphorylation of this tyrosine in the cytoplasmic domain of the FcR is sufficient to promote the interaction between the receptor and Ship. Affinity precipitates from cells stimulated with F(ab′)2 or intact anti-IgG did not significantly change the ability of the phosphorylated peptide to interact with Ship. This indicates that phosphorylation of an intermediary protein such as Shc is not essential for the interaction.
      Ship catalyzes the hydrolysis of the 5-phosphate of inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ). Our data indicate that interaction of this enzyme with the antigen receptor complex, through its association with the phosphorylated FcR, is likely to alter locally the metabolism of either inositol phosphates or phosphatidylinositol phosphates. This may directly regulate the activity of the plasma membrane calcium channel. It is interesting to note that retroviral expression of Ship in FD-fms cells results in strong inhibition of growth (
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold A.
      • Rohrschneider L.R.
      ). This inhibition may be due to the alteration of the ability of cells to mobilize calcium.
      In contrast to the effect on FcR, Ship, and Shc, co-ligation of sIg and FcR resulted in the decrease of phosphorylation of several proteins, including CD19 and other, as yet uncharacterized, proteins. Activation of cells through the antigen receptor alone results in an increase in the tyrosine phosphorylation of both CD19 and CD22 (
      • Chalupny N.J.
      • Kanner S.B.
      • Schieven G.L.
      • Wee S.F.
      • Gilliland L.K.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Roifman C.M.
      • Ke S.
      ,
      • Schulte R.J.
      • Campbell M.-A.
      • Fischer W.H.
      • Sefton B.M.
      ,
      • Peaker C.J.G.
      • Neuberger M.S.
      ). In our experiments, co-ligation of the FcR to sIg greatly decreased the phosphorylation state of CD19 but did not significantly alter the level of phosphorylation of CD22. Several signaling molecules are found associated with CD19 following activation of B cells through the antigen receptor; these include PI3-kinase, Vav, and Fyn (
      • Weng W.K.
      • Jarvis L.
      • LeBien T.W.
      ). Our results show that on co-cross-linking the antigen and Fc receptors, the reduction in tyrosine phosphorylation of CD19 gave rise to a corresponding decrease in the level of associated PI3-kinase. This suggests that PI3-kinase activity in the receptor complex may play a crucial role in the regulation of influx of extracellular calcium. Addition of wortmannin to the cells prior to activation with F(ab′)2 anti-IgG markedly abrogated the secondary influx of extracellular calcium although having only a modest effect on the mobilization of calcium from intracellular stores. The inhibitor also induced a rapid decrease in the levels of intracellular calcium in cells that were already mobilizing extracellular calcium and blocked sIg-induced IL-2 production. Wortmannin inhibits PI3-kinase activity at concentrations down to 10−8M (
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Powis G.
      • Bonjouklian R.
      • Berggren M.M.
      • Gallegos A.
      • Abraham R.
      • Ashendel C.
      • Zalkow L.
      • Matter W.F.
      • Dodge J.
      • Grindley G.
      • Vlahos C.J.
      ,
      • Arcaro A.
      • Wymann M.P.
      ); therefore, its effect on the mobilization of calcium is consistent with (but not proof of) PI3-kinase activity, in association with CD19, playing a role in the regulation of the calcium channel.
      Analysis of cells that were stimulated initially through sIg and then subsequently restimulated by co-ligation of sIg and the FcR indicated that the changes in tyrosine phosphorylation of the proteins occurred rapidly. The kinetics of these biochemical changes are of the right time frame to be able to account for the changes in influx of extracellular calcium, which diminishes over the first few minutes following co-ligation of the receptors. It should be pointed out that the effect of intact anti-Ig on tyrosine phosphorylation of all of these proteins was not simply due to the abrogation of an influx of extracellular calcium. Removal of extracellular calcium with 2.5 mM EGTA did not alter the tyrosine phosphorylation state of any of the proteins when the cells were activated with F(ab′)2 anti Ig (data not shown).
      In summary, co-cross-linking of the FcR to sIg gives rise to a very discrete set of changes in signal transduction. Many of the signaling pathways that were stimulated following ligation of the antigen receptor alone were unaltered when sIg was co-cross-linked to the FcR. These include the activation of several different protein kinases, the phosphorylation of many intracellular proteins, and the activation of the transcription factor NFκB (
      • Tepper M.A.
      • Nadler S.G.
      • Esselstyn J.M.
      • Sterbenz K.G.
      ) (data not shown). Thus the role of SHP-1 in the negative regulation of B cell receptor signaling is very specific and is not a global shutdown of tyrosine phosphorylation at the antigen receptor. Our data suggest that dephosphorylation of CD19 (perhaps by SHP-1) and the subsequent loss of association of PI3-kinase are crucial steps in altering calcium mobilization. This may be mimicked by the inhibition of PI3-kinase activity with wortmannin. In addition, our studies have shown that the phosphorylation of the FcR promotes its interaction with Ship, an inositol 5′-phosphatase. Thus co-ligation of the FcR and sIg markedly changes the overall activity of lipid-modifying enzymes that are associated with the receptor complex. In particular, it is possible that the local levels of phosphatidylinositol 3,4,5-trisphosphate are critical to the regulation of opening and closing of the calcium channel.

      Acknowledgments

      We thank Dr. Bruce Cohen (Bristol-Myers Squibb) for critical reading of the manuscript and helpful discussions and Dr. Joe Bolen (DNAX) for providing antibodies.

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