The dynamics of protein kinase B regulation during B cell antigen receptor engagement

Abstract

This study has used biochemistry and real time confocal imaging of green fluorescent protein (GFP)-tagged molecules in live cells to explore the dynamics of protein kinase B (PKB) regulation during B lymphocyte activation. The data show that triggering of the B cell antigen receptor (BCR) induces a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of activated B cells. Hence, PKB has three potential sites of action in B lymphocytes; transiently after BCR triggering PKB can phosphorylate plasma membrane localized targets, whereas during the sustained B cell response to antigen, PKB acts in the nucleus and the cytosol. Membrane translocation of PKB and subsequent PKB activation are dependent on BCR activation of phosphatidylinositol 3-kinase (PI3K). Moreover, PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes. However, under conditions of continuous PI3K activation or BCR triggering there is only transient recruitment of PKB to the plasma membrane, indicating that there must be a molecular mechanism to dissociate PKB from sites of PI3K activity in B cells. The inhibitory Fc receptor, the FcgammaRIIB, mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase SHIP into the BCR complex. Herein we show that coligation of the BCR with the inhibitory FcgammaRIIB prevents membrane targeting of PKB. The FcgammaRIIB can thus antagonize BCR signals for PKB localization and prevent BCR stimulation of PKB activity which demonstrates the mechanism for the inhibitory action of the FcgammaRIIB on the BCR/PKB response.

Figure 2

PKB localization in B cells. (A) Confocal images of live A20 cells expressing GFP-tagged full length PKB (top) or GFP-tagged PKB-PH domain (bottom). Cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images taken at 10-s intervals. (B) A20 cells were stimulated for the indicated times at 37°C with 10 μg/ml F(ab′)₂ fragment of anti– mouse IgG which triggers the BCR. The data show Western blots of total cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera and phosphospecific GSK3α and pan GSK3α antisera.

Figure 2

PKB localization in B cells. (A) Confocal images of live A20 cells expressing GFP-tagged full length PKB (top) or GFP-tagged PKB-PH domain (bottom). Cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images taken at 10-s intervals. (B) A20 cells were stimulated for the indicated times at 37°C with 10 μg/ml F(ab′)₂ fragment of anti– mouse IgG which triggers the BCR. The data show Western blots of total cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera and phosphospecific GSK3α and pan GSK3α antisera.

Figure 1

BCR and FcγRIIb regulation of PKB. The data show PKB phosphorylation and activity in quiescent A20 cells or A20 cells stimulated for 10 min at 37°C with the following stimuli: 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR; 15 μg/ml of intact IgG of anti–mouse IgG which coligates the BCR and the FcγRIIB complex; 15 μg/ml of intact Ig in cells pretreated for 30 min with 2.5 μg/ml anti-FcγRIIB which prevents FcγRIIB/BCR coligation and allows intact Ig to trigger the BCR. The data in A show Western blots of total cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera. The data in B show PKB kinase activity of immunoprecipitated Ha epitope–tagged PKB measured using histone H2B as a substrate. H2B substrate phosphorylation was analyzed by autoradiography. The data in C show Western blots of total cell lysates performed with phosphospecific GSK3α and pan GSK3α antisera.

Figure 3

Cellular localization of PKB in B cells. The data show Western blot analyses of nuclear and cytosolic extracts of A20 cells performed with serine 473 phosphospecific PKB antibody and pan PKB antisera or an antisera reactive with the serine/threonine kinase PKD/PKCμ. Cells were either quiescent or stimulated for the indicated times with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG.

Figure 4

BCR regulation of PKB membrane localization and activity is dependent on PI3K. (A) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images were taken at 10-s intervals. The data show confocal images of four cells at the zero and 15-s time points. (B) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images taken at 5-s intervals. The data show confocal images of two cells at the zero and 15-s time points. (C) A20 cells were untreated or pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG for 5 min. The data show Western blot analyses of A20 cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera.

Figure 4

BCR regulation of PKB membrane localization and activity is dependent on PI3K. (A) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images were taken at 10-s intervals. The data show confocal images of four cells at the zero and 15-s time points. (B) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images taken at 5-s intervals. The data show confocal images of two cells at the zero and 15-s time points. (C) A20 cells were untreated or pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG for 5 min. The data show Western blot analyses of A20 cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera.

Figure 4

BCR regulation of PKB membrane localization and activity is dependent on PI3K. (A) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images were taken at 10-s intervals. The data show confocal images of four cells at the zero and 15-s time points. (B) Confocal images of live A20 cells expressing GFP-tagged full length PKB. Cells were pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR and confocal images taken at 5-s intervals. The data show confocal images of two cells at the zero and 15-s time points. (C) A20 cells were untreated or pretreated for 30 min with 5 μM of Ly294002, the PI3K inhibitor, and then stimulated with 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG for 5 min. The data show Western blot analyses of A20 cell lysates performed with serine 473 phosphospecific PKB antibody and pan PKB antisera.

Figure 5

PI3K signals are sufficient to activate PKB in B cells. (A) A20 cells were transfected with either 5, 10, or 20 μg of pEF rCD2p110 (active PI3K) or the equivalent amounts of the control catalytically inactive pEF rCD2p110R/P mutant. Cells were maintained in culture for 14 h. The data show Western blot analysis of total cell lysates from control cells or cells expressing the PI3K mutant. The top shows Western blot experiments with the rat CD2 antibody OX34 which monitors cellular levels of the rCD2 PI3K chimeras. The middle and bottom show, respectively, Western blot analyses with serine 473 phosphospecific PKB antibody and pan PKB antisera. (B) A20 cells were transfected with 20 μg of pEF rCD2p110 (active PI3K). Cells were maintained in culture for 14 h. The data show Western blot analysis of total cell lysates from cells expressing rCD2p110 or after 3 h of incubation with 5 μM of Ly294002. The data show Western blot analyses with serine 473 phosphospecific PKB antibody (top) and pan PKB antisera (bottom).

Figure 5

PI3K signals are sufficient to activate PKB in B cells. (A) A20 cells were transfected with either 5, 10, or 20 μg of pEF rCD2p110 (active PI3K) or the equivalent amounts of the control catalytically inactive pEF rCD2p110R/P mutant. Cells were maintained in culture for 14 h. The data show Western blot analysis of total cell lysates from control cells or cells expressing the PI3K mutant. The top shows Western blot experiments with the rat CD2 antibody OX34 which monitors cellular levels of the rCD2 PI3K chimeras. The middle and bottom show, respectively, Western blot analyses with serine 473 phosphospecific PKB antibody and pan PKB antisera. (B) A20 cells were transfected with 20 μg of pEF rCD2p110 (active PI3K). Cells were maintained in culture for 14 h. The data show Western blot analysis of total cell lysates from cells expressing rCD2p110 or after 3 h of incubation with 5 μM of Ly294002. The data show Western blot analyses with serine 473 phosphospecific PKB antibody (top) and pan PKB antisera (bottom).

Figure 6

PKB localization in cells expressing constitutively active PI3K. (A) Confocal images of live A20 cells cotransfected with 20 μg of pEF rCD2p110 (active PI3K) and either GFP-tagged full length PKB (left) or GFP-tagged PKB-PH domain (right). After transfection, cells were maintained in culture for 14 h before confocal imaging. (B) Confocal images of live A20 cells cotransfected with 20 μg of pEF rCD2p110 (active PI3K) and GFP-tagged PKB-PH domain. After transfection, cells were maintained in culture for 14 h before confocal imaging. Confocal images were taken at 10-s intervals after addition of 5 μM of Ly294002 to inhibit PI3K activity.

Figure 7

The FcγRIIb prevents membrane translocation of PKB. Confocal images of live A20 cells expressing GFP-tagged full length PKB. A20 cells stimulated at 37°C with the following stimuli: 10 μg/ml F(ab′)₂ fragment of anti–mouse IgG which triggers the BCR; 15 μg/ml of intact Ig which coligates the BCR and the FcγRIIB complex; and 15 μg/ml of intact Ig in cells pretreated for 30 min with 2.5 μg/ml anti-FcγRIIB which prevents FcγRIIB/ BCR coligation and allows intact Ig to trigger the BCR. Confocal images were taken at 5-s intervals; the data show confocal images taken 15 s after A20 cell stimulation.