B Cell Antigen Receptor-Mediated Activation of Cyclin-Dependent Retinoblastoma Protein Kinases and Inhibition by Co-Cross-Linking with Fcγ Receptors

Reagents

Protein A/G PLUS-agarose, rabbit IgG for mouse Cdk4 (sc-260), anti-human p27^Kip1 Ab (sc-528), anti-cyclin E Ab (sc-481), anti-rabbit and anti-mouse IgG-HRP Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human Cdk2 Ab (06–505) was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-FcγR mAb (clone 2.4G2) and human pRb mAb (clone G3-245) and anti-mouse p21 Ab (13436E) were obtained from PharMingen (San Diego, CA). The anti-pRb (Ser⁷⁸⁰) Ab was from MBL International (Watertown, MA). The production of mouse anti-human cyclin D2 Ab (DCS-3 and DCS-5) and anti-human cyclin D3 Ab (DCS-22) have been described (51). Anti-actin Ab (A-2066) was obtained from Sigma (St. Louis, MO) and anti-human Cdk7 mAb (KAP-CC010) and Cdk7/MO15 peptide (aa 328–346) were purchased from StressGen Biotechnologies (Victoria, BC, Canada). Whole rabbit anti-mouse IgG, F(ab′)₂ fragments of rabbit anti-mouse IgG, and anti-mouse IgM was obtained from Cappel/ICN Biomedicals (Aurora, OH). Enhanced chemiluminescent reagents were from Kirkegaard & Perry (Gaithersburg, MD). Affinity-purified Rb protein corresponding to aa 769–921 (sc-4112) was from Santa Cruz Biotechnology, and truncated p56 Rb was from QED Advanced Research Technologies (San Diego, CA). F(ab′)₂ fragments of anti-mouse μ were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Protein A-agarose was obtained from Life Technologies (Gaithersburg, MD). Dr. Mark Solomon (Yale University, New Haven, CT) kindly provided the GST-cyclin A and HA-Cdk2 expression plasmids.

B cell isolation

BALB/c mice at 8–12 wk were obtained from Taconic Laboratories (Germantown, NY) and housed at Boston College. Mice were cared for and handled at all times in accordance with National Institutes of Health and institutional guidelines. Mature B lymphocytes were isolated from spleens by depletion of T cells with anti-Thy-1.2 plus rabbit complement (Accurate Chemical and Scientific, Westbury, NY) (52); macrophages (and other adherent cells) were removed by plastic adherence. RBC and nonviable cells were removed by sedimentation on Lympholyte M (Accurate Chemical and Scientific). Small resting B cells were selected by Percoll gradient centrifugation as described by DeFranco et al. (1). The resulting B cells were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10 mM HEPES (pH 7.5), 2 mM l-glutamine, 5 × 10⁻⁵ M 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml Fungizone, and 10% heat-inactivated FCS (BioWhittaker). B cells were maintained in a 37°C humidified incubator at 5% CO₂.

Proliferation assay

B cells (2 × 10⁵/well) were cultured in 96-well flat-bottom microtiter plates in the presence and absence of stimuli as indicated in the figure legends. Before the indicated times, cells were pulsed for 3 h with 0.5 μCi [³H]uridine (39.7 Ci/mmol, New England Nuclear, Boston, MA) and 6 h with 0.5 μCi [³H]thymidine (20 Ci/mmol, New England Nuclear). Cells were harvested onto glass fiber filters and quantitated by liquid scintillation spectrophotometry.

Immunoblot analysis

B lymphocytes were solubilized in 100 μl of solubilization buffer (50 mM HEPES (pH 7.4), 1.5 mM EGTA, 137 mM NaCl, 15 mM MgCl₂, 0.1% Triton X-100, 75 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM PMSF, and 1 μg/ml aprotinin/leupeptin) (8, 48). Insoluble material was removed by centrifugation at 15,000 × g for 15 min and protein content was quantitated with Bradford reagents (Bio-Rad Laboratories, Hercules, CA). Cellular protein was separated through a 10–12% polyacrylamide SDS gel and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The Immobilon-P membrane was incubated for 6 h at room temperature in TBST (20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk. After washing the membrane with TBST (twice for 5 min), the membrane was incubated for 60 min to 18 h (4°C) with primary Ab as specified in the figure legends. The membrane was then washed several times with TBST and incubated with an anti-rabbit or anti-mouse IgG-conjugated HRP at 1:2000 in TBST for 90 min. The membrane was washed several times with TBST and developed with enhanced chemiluminescent reagent. Where indicated, autoradiograms were analyzed by densitometry using a Molecular Dynamics Personal Densitometer equipped with ImageQuant software (Sunnyvale, CA).

Immunoprecipitation

Nondenaturing extracts were prepared from 10⁷ B cells according to the method described by Zhang et al. (44). Cells were lysed by incubation for 60 min (4°C) in ice-cold Nonidet P-40 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 25 μg/ml leupeptin/aprotinin, 1 mM Na₃VO₄, and 10 mM β-glycerophosphate) (8). Insoluble material was removed by centrifugation at 15,000 × g for 15 min (4°C). Cell lysates were then incubated for 5 h with 1.5 μg of the indicated Abs, followed by the addition of 50 μl of a 1:1 slurry of protein A-agarose. The resulting immune complexes were collected and separated through a 10% polyacrylamide-SDS gel. The proteins were transferred to Immobilon-P membrane and immunoblotted as described above.

Rb kinase assays

B cells were sonicated in Rb buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20, and 10% glycerol) containing 1 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin/aprotinin, 1 mM NaF, and 1 mM Na₃VO₄ (53). Insoluble material was removed by centrifugation and the resulting supernatants were incubated with 1.5 μg nonimmune rabbit IgG, 1.5 μg anti-Cdk4, anti-Cdk6, or anti-Cdk2 Abs for 3 h at 4°C. After 3 h, the supernatants were incubated with 50 μl of a 1:1 slurry of protein G/A-agarose and recovered by centrifugation (15,000 × g for 1 min). The immune complexes were washed six times with Rb buffer, three times in a buffer containing 50 mM HEPES (pH 7.4), 5 μM ATP, and 1 mM DTT, and then resuspended in 30 μl Rb kinase buffer (50 mM HEPES (pH 7.5), 5 mM MnCl₂, 10 mM MgCl₂, and 1 mM DTT) containing 5 μM ATP, 10 μCi [γ-³²P]ATP (6000 Ci/mmol, New England Nuclear) and 1 μg purified recombinant Rb substrate. The kinase assays were terminated after 30 min at 30°C by the addition of 2× SDS sample buffer, and the reaction products were separated through a 10% polyacrylamide-SDS gel. Phosphorylated Rb was detected by autoradiography of the dried gel.

Cdk-activating kinase (CAK) assays

Production and purification of HA-Cdk2 and GST-cyclin A fusion proteins from Escherichia coli strain BL21(DE3) were performed as described by Connell-Crowley et al. (54). B cells were prepared by sonication at 4°C in CAK lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 1 mM PMSF, and 1 μg/ml leupeptin/aprotinin). The cell lysates were incubated for 3 h with 2.5 μg anti-Cdk7 mAb/10⁷ B cells, and the immune complexes were recovered by incubation with 30 μl protein A/G PLUS agarose (90 min) (55, 56, 57). The immune complexes were washed five times in CAK lysis buffer, twice in Rb kinase buffer, and then resuspended in 25 μl Rb kinase buffer containing 0.5 μM ATP, 5 μCi [γ-³²P]ATP (6000 Ci/mmol, New England Nuclear), 1 μg GST-cyclin A, and 3 μg of HA-Cdk2 as substrate. The reactions were terminated after 15 min (30°C) by the addition of 2× SDS sample buffer and separated through a 12% polyacrylamide-SDS gel. Phosphorylated HA-Cdk2 was detected by autoradiography of the dried gel.

Mature B cells exhibit reduced BCR-stimulated RNA and DNA synthesis in response to co-cross-linking FcγR

To evaluate the effect of BCR-FcγR co-cross-linking on cell cycle entry, splenic B lymphocytes were subjected to Percoll density sedimentation to isolate quiescent B cells and then stimulated with 10 μg/ml F(ab′)₂ anti-Ig or 10 μg/ml whole anti-Ig. B cells stimulated with F(ab′)₂ anti-Ig exhibited a 3.4-fold increase in [³H]uridine incorporation compared with control B cells cultured in medium alone (Table I⇓). Support for F(ab′)₂ anti-Ig-induced cell cycle progression to S phase was demonstrated by increased [³H]thymidine incorporation at 24 h and 48 h (18-fold and 49-fold above control B cells, respectively). By contrast, B cells cultured with whole anti-Ig for 24 h incorporated [³H]uridine and [³H]thymidine at levels equal to control B cells; these cells exhibited a relatively low level of [³H]thymidine incorporation at 48 h (3.2-fold above control B cells). Further support for inhibition of B cell activation by whole anti-Ig was obtained from flow cytometry in which B cells failed to undergo increased cell size, whereas B cells stimulated with F(ab′)₂ anti-Ig were enlarged at 24 h relative to control B cells, as monitored by forward scattering (data not shown).

It should be noted that B cells cultured in the presence of 10 μg/ml whole anti-Ig and 20 μg/ml anti-FcγR mAb (2.4G2), an immunologic reagent that binds FcγR and blocks IgG-FcγR binding (14), exhibited increased [³H]thymidine incorporation that was comparable to F(ab′)₂ anti-Ig, indicating that inhibition of proliferation by whole anti-Ig was mediated by FcγR (data not shown). Collectively, these findings are consistent with several studies demonstrating inhibition of G₁-to-S progression by co-cross-linking the BCR to FcγR (12, 13, 14, 15). Of note, in subsequent experiments the analyses of B cells treated with whole anti-Ig was not extended beyond 31 h due to decreased cell viability and detection of apoptotic cells; B cells stimulated with F(ab′)₂ anti-Ig exhibited >90% viability through 48 h.

BCR-induced Rb protein phosphorylation is blocked by coengagement of FcγR

The mechanism underlying inhibition of BCR-stimulated G₁-to-S progression by FcγR remains incompletely defined. Rb functions to suppress cell growth and is expressed in a hypophosphorylated state in quiescent mammalian cells (28). Hyperphosphorylation of Rb during G₁ phase, a modification that results in its functional inactivation, leads to S phase entry (28, 29, 30). We therefore investigated the regulation of Rb phosphorylation by F(ab′)₂ anti-Ig and whole anti-Ig. To monitor Rb phosphorylation, whole cell lysates prepared from B cells were separated by SDS-PAGE and immunoblotted with an anti-human pRb mAb. Previous studies have established the specificity of this assay for monitoring Rb phosphorylation (56). The existence of multiple Rb forms during SDS-PAGE is indicative of Rb phosphorylation with hyperphosphorylated Rb exhibiting a greater apparent m.w. in comparison to hypo- or unphosphorylated Rb. Rb was expressed as a single band of ∼110 kDa in quiescent B cells (Fig. 1⇓A). Stimulation of resting B cells with F(ab′)₂ anti-Ig led to the appearance of a second slower migrating Rb form at the 12-h time point (Fig. 1⇓A, indicated by arrow/pRb); this band was more prominent at 24 h and 31 h. The appearance of the second M_r Rb form parallels the timing of G₁-to-S progression (1, 2). In the same experiment, Rb in lysates from B cells treated with whole anti-Ig was expressed as a single band on SDS-polyacrylamide gels and exhibited a M_r similar to resting B cells. We conclude that quiescent B cells stimulated to progress to S phase by F(ab′)₂ anti-Ig exhibit a time-dependent increase in Rb phosphorylation, whereas signals derived from FcγR prevent BCR-induced Rb phosphorylation.

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FIGURE 1.

Inhibition of BCR-induced Rb phosphorylation by B lymphocytes in response to BCR-FcγR co-cross-linking. Resting B cells were cultured in medium alone (lane M) or stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig) or 10 μg/ml whole anti-Ig (wIg) for the indicated time in hours. Cells were then collected and detergent solubilized protein lysates (20 μg) were separated by SDS-PAGE and transferred to Immobilon-P. A, Immobilon-P was immunoblotted with 1 μg/ml anti-human pRb mAb that recognizes both hypo- and hyperphosphorylated Rb. Arrows indicate position of Rb (Rb) and BCR-induced Rb phosphorylation (pRb). B, Immobilon-P was immunoblotted with 1 μg/ml anti-pRb(Ser⁷⁸⁰) Ab. Arrow indicates position of phospho(Ser⁷⁸⁰)Rb. The m.w. of standard proteins (kDa) is indicated on the left.

Evidence supporting the notion that BCR-FcγR co-cross-linking blocks phosphorylation of Rb by cyclin D holoenzyme complexes was obtained using an anti-pRb Ser⁷⁸⁰ Ab that recognizes the Ser⁷⁸⁰ phosphorylated form of Rb. Cyclin D-Cdk4/6 complexes in vitro phosphorylate Ser⁷⁸⁰ (33). In vivo this site is phosphorylated in early G₁ phase, concomitant with the expression and activation of Cdk4 (33). Although cyclin D-Cdk4/6 complexes target additional sites on Rb, Ser⁷⁸⁰ phosphorylation of endogenous Rb should be indicative of the presence of in vivo activated cyclin D-Cdk4 or -Cdk6 complexes. Ser⁷⁸⁰ phosphorylation was initially detected in lysates from F(ab′)₂ anti-Ig stimulated B cells at the 12-h time point and increased to maximal levels at ∼31 h, declining thereafter. By comparison, extracts from quiescent and whole anti-Ig treated B cells were devoid of detectable Rb Ser⁷⁸⁰ phosphorylation at each time point examined (Fig. 1⇑B).

G₁-Cdks from B lymphocytes treated with whole anti-Ig do not support in vitro Rb protein phosphorylation

Abundant evidence supports the role of Cdk4/6 and Cdk2 in attenuating the growth inhibitory function of Rb via phosphorylation (30, 31, 32, 33, 34). In mature B cells, Cdk4 and Cdk6 are rapidly up-regulated in response to F(ab′)₂ anti-Ig stimulation, whereas Cdk2 exhibits a delayed expression profile, occurring near the G₁/S transition (8, 9, 47, 48). Because cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes contribute to the phosphorylation of Rb following B cell stimulation, we considered the possibility that they might be targets of biochemical signals derived from BCR-FcγR co-cross-linking. We therefore measured Cdk4/6- and Cdk2-dependent Rb kinase activities in lysates prepared from B cells under growth inhibitory and stimulatory conditions. Control B cells were essentially devoid of Cdk4/6 and Cdk2 activities, as measured by in vitro immune complex kinase assays with recombinant Rb protein as substrate (Fig. 2⇓, lanes M). F(ab′)₂ anti-Ig stimulation of B cells led to activation of Cdk4 that was detected at 12 h, increased at 24 h and 31 h, and then declined at 48 h (Fig. 2⇓, F(ab′)₂ anti-Ig, lanes Cdk4). Cdk6 immune complexes from BCR-stimulated B cells also supported Rb phosphorylation with a similar activation profile (Fig. 2⇓, F(ab′)₂ anti-Ig, lanes Cdk6). Cdk2 exhibited a delayed Rb phosphorylation profile following BCR cross-linking, in keeping with previous reports (Fig. 2⇓, F(ab′)₂ anti-Ig, lanes Cdk2) (8, 9, 48). By contrast, in several experiments we did not detect significant levels of Cdk4- or Cdk6-dependent Rb kinase activity from whole anti-Ig-treated B cells at the time points examined (Fig. 2⇓, whole anti-Ig). Moreover, Cdk2-immune complexes from BCR-FcγR co-cross-linked B cells were also devoid of Rb kinase activity. It should be noted that B cells precipitated with nonimmune rabbit serum were devoid of measurable Rb kinase activity (Fig. 2⇓, NRS). Collectively, these data suggest that maintenance of endogenous Rb in a hypo- or unphosphorylated state following BCR-FcγR coligation results from a failure to activate the cellular pool of known G₁-Cdks.

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FIGURE 2.

G₁-Cdk-mediated Rb protein phosphorylation in BCR-stimulated B cells is blocked by FcγR co-cross-linking. B lymphocytes were cultured in medium alone (lane M) or stimulated with 10 μg/ml F(ab′)₂ anti-Ig or whole anti-Ig for the indicated time in hours. Cell lysates were then prepared and immunoprecipitated (IP) with either 1.5 μg control rabbit sera (NRS), anti-Cdk4, anti-Cdk6, or anti-Cdk2 Abs. Immune complex kinase activity was then assayed in vitro using a truncated Rb protein substrate as described in Materials and Methods. Arrows indicate position of pRb.

Co-cross-linking BCR-FcγR blocks up-regulation of cyclin E and Cdk2 protein levels, but does not affect BCR-induced D-type cyclin and Cdk4/6 protein expression

Given the above data, we postulated that FcγR may abrogate G₁-Cdks and/or cyclin expression and in this way achieve its inhibitory effect on Cdk4/6 and Cdk2 activities. To test this possibility, B cells were evaluated for expression of G₁-Cdks and their associated cyclin subunits. Stimulation of B cells with F(ab′)₂ anti-Ig led to an increased cellular level of Cdk4 at 12 h, which was maintained at each subsequent time point examined (Fig. 3⇓A). Similarly, whole anti-Ig treatment of B cells led to a time-dependent increase in Cdk4 levels. Cdk4 in lysates from F(ab′)₂ anti-Ig stimulated B cells migrated in SDS-polyacrylamide gels as two distinct bands. Both bands were judged specific insofar as preincubation of the anti-Cdk4 Ab with Cdk4 antigenic peptide resulted in a significant reduction in immunoreactivity. The nature of the faster migrating M_r form is presently unknown but may represent a BCR-dependent posttranslational modification of Cdk4, which is prevented by FcγR co-cross-linking.

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FIGURE 3.

G₁-Cdk expression in BCR-stimulated and BCR-FcγR-inhibited B cells. B lymphocytes were cultured in medium alone (lane M) or stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig) or whole anti-Ig (wIg) for the indicated time in hours (12, 24, 31 h). A, Cell lysates were prepared, and 10 μg protein was separated by SDS-PAGE and immunoblotted with 1.5 μg/ml anti-Cdk4 Ab. pC4 denotes immunoblotting with anti-Cdk4 Ab preincubated with 5 μg antigenic Cdk4 peptide; the extract was prepared from B cells stimulated with F(ab′)₂ anti-Ig. The Immobilon-P membrane was stripped with 10 mM glycine (pH 2.0) plus 1% SDS and immunoblotted with 1.5 μg/ml anti-Cdk6 Ab, and then with a 1:2000 dilution of anti-actin Ab. B, Extracts were separated by SDS-PAGE and immunoblotted with 1.5 μg/ml anti-Cdk2 Ab. pC2 denotes immunoblotting of the 31 h F(ab′)₂ anti-Ig extract with anti-Cdk2 Ab preincubated with 5 μg antigenic Cdk2 peptide. The m.w. of standard proteins (kDa) is indicated on the left.

Cdk6 was detected in quiescent B cells and its level increased at 12 h following BCR cross-linking; the heightened level of Cdk6 were maintained through 31 h (Fig. 3⇑A). The level of Cdk6 in whole anti-Ig-treated B cells at 12 h was equal to quiescent B cells and exhibited a modest increase at 24 h and 31 h. To ensure that equal amount of protein was immunoblotted in each lane, the blot was stripped and actin levels measured by immunoblotting with an anti-actin Ab (Fig. 3⇑A). Interestingly, antiserum to p33Cdk2 did not detect expression of Cdk2 in extracts from control and whole anti-Ig-treated B cells at the time points examined, whereas F(ab′)₂ anti-Ig led to a time-dependent increase in Cdk2 protein levels, initially detected at 24 h (Fig. 3⇑B).

Low levels of cyclins D2 and D3 expression were detected in lysates from control B cells (Fig. 4⇓A); whole anti-Ig treatment of resting B cells led to a time-dependent increase in cyclins D2 and D3 levels with maximal expression at 24 h and 31 h, respectively. As expected, the levels of cyclins D2 and D3 were up-regulated in BCR-stimulated B cells at 24 h, in agreement with a more detailed time course analyses previously reported (8, 9, 47). Using antisera specific for cyclin E, we detected two relatively faint bands at approximate m.w. of 55 kDa in control B cells (Fig. 4⇓B, denoted by arrows). Both bands were blocked by preincubation of the anti-cyclin E Ab with immunizing peptide before immunoblotting and thus, determined to be specific (Fig. 4⇓B, lanes pcE). The level of the immunoreactive bands detected in control B cells did not appear to significantly change in whole anti-Ig-treated B cells. By contrast, expression of both cyclin E bands in F(ab′)₂ anti-Ig stimulated B cells were elevated in a time-dependent manner. The level of actin in each lane of Fig. 4⇓B was equal as determined by stripping the blot and immunoblotting with anti-actin Ab (data not shown).

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FIGURE 4.

G₁-cyclin expression in BCR-stimulated and BCR-FcγR-inhibited B cells. B lymphocytes were cultured in medium alone (lane M) or stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig) or 10 μg/ml whole anti-Ig (wIg) for the indicated time in hours. A, Cell lysates were then prepared and 10 μg of protein was separated by SDS-PAGE and immunoblotted with anti-cyclin D2 or anti-cyclin D3 mAbs (1:500 dilution of hybridoma supernatants). The Immobilon-P membrane was stripped and reprobed with anti-actin Ab. B, Lysate protein (20 μg) was analyzed by SDS-PAGE and immunoblotted with 1.5 μg/ml anti-cyclin E Ab. pcE denotes immunoblotting of the 31 h F(ab′)₂ anti-Ig extract with anti-cyclin E Ab preincubated with 5 μg antigenic cyclin E peptide. Arrows indicate the position of the immunoreactive cyclin E doublet.

We conclude from these data that the lack of Cdk2 protein expression following BCR-FcγR co-cross-linking suffices in explaining the inability of Cdk2-immune complexes to support in vitro Rb phosphorylation. Of note, it is not known if the level of cyclin E expression in whole anti-Ig-treated B cells is below a threshold level necessary to otherwise activate Cdk2. In contrast, individual components comprising cyclin D-Cdk4/6 complexes are up-regulated in response to BCR-FcγR co-cross-linking, suggesting that inhibition of Cdk4/6 kinase activities by FcγR is mediated at a posttranslational level.

CAK activity is not affected by cross-linking BCR-FcγR

Apart from binding regulatory cyclin, posttranslational modification of Cdks is a focal point for regulating their kinase activity. In particular, phosphorylation on a single threonine residue (Thr¹⁶⁰ in Cdk2 and Thr¹⁷² in Cdk4) by CAK is essential for activity (55, 57). CAK consists of Cdk7, cyclin H, and the p36 MAT1 assembly factor (58). It is possible that CAK might be inhibited as a consequence of BCR-FcγR coligation, thereby preventing activation of Cdk4 and Cdk6. Therefore, we sought to develop an in vitro assay to measure CAK activity in primary B lymphocytes. B cells were solubilized in a 0.1% Tween 20 buffer to maintain CAK and CAK-like activities, and then Cdk7 was immunoprecipitated using an anti-human Cdk7 mAb (54, 55, 57). The resulting immune complexes were assayed in vitro for phosphorylation of HA-Cdk2 substrate based on the method of Connell-Crowley et al. (54). In preliminary experiments, we found that both Cdk7 (Fig. 5⇓A) and cyclin H (Fig. 5⇓B) were expressed in control, whole anti-Ig, and F(ab′)₂ anti-Ig stimulated B cells. (Note that only a single time point is shown in Fig. 5⇓, A and B; however, the levels of Cdk7 and cyclin H did not change over time through 31 h and 48 h following BCR-FcγR coligation and BCR cross-linking, respectively (data not shown). Cdk7-mediated phosphorylation of HA-Cdk2 substrate was observed in lysates from control, whole anti-Ig, and F(ab′)₂ anti-Ig stimulated B cells (Fig. 5⇓C). The specificity of this assay was confirmed in several control experiments. 1) HA-Cdk2 was not efficiently phosphorylated by nonimmune IgG immunoprecipitates in comparison to anti-Cdk7 immunoprecipitates (compare Fig. 5⇓D, lanes A and B, respectively). 2) Preincubating the anti-Cdk7 mAb with immunizing Cdk7/MO15 peptide before immunoprecipitation significantly reduced phosphorylation of HA-Cdk2 substrate by immune complexes (Fig. 5⇓D, lane C). 3) No anti-Cdk7 immune complex-mediated phosphorylation was observed from in vitro kinase assays devoid of added HA-Cdk2 substrate (Fig. 5⇓D, lane D) or in assays containing HA-Cdk2 substrate devoid of GST-cyclin A (Fig. 5⇓D, lane E). It is noteworthy that the autoradiogram was exposed for an extended period of time to detect any low level phosphorylation. We conclude that FcγR-derived signals do not significantly affect CAK activity.

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FIGURE 5.

CAK activity is not affected by BCR-FcγR co-cross-linking. B cells were cultured in medium alone (lane M), stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig), or whole anti-Ig (wIg) for 24 h. Cell lysates were then immunoblotted with: 1.5 μg/ml anti-Cdk7 Ab (A) or 1.5 μg/ml anti-cyclin H Ab (B). In A, lane pC7 denotes immunoblotting with anti-Cdk7 Ab preincubated with 5 μg antigenic MO15/Cdk7 peptide; lane + denotes 10 μg mouse brain extract which is a source for Cdk7. C, B cells were cultured as described above for the indicated time in hours. Nonimmune mouse IgG (NI) and anti-Cdk7 immunoprecipitates were recovered and assayed in vitro for phosphorylation of HA-Cdk2 substrate as described in Materials and Methods. D, Lysates from control B cells were immunoprecipitated with 2.5 μg/ml control sera (lane A), 2.5 μg/ml anti-Cdk7 Ab in the absence (lane B), and in the presence of 5 μg antigenic MO15/Cdk7 peptide (lane C). The resulting immune complexes were assayed for HA-Cdk2 phosphorylation. Lane D represents Cdk7 immune complex kinase reactions in the absence of HA-Cdk2 substrate. Lane E denotes Cdk7 immune complex kinase reactions containing HA-Cdk2 substrate, but devoid of GST-cyclin A. Note that the autoradiogram was exposed for an extended period of time to detect low level phosphorylation. Arrows in C and D indicate phosphorylated HA-Cdk2. The m.w. of standard proteins (kDa) is indicated on the left.

FcγR blocks stable cyclin D-Cdk4/6 holoenzyme complex formation

We next sought to examine the possibility that inhibition of Cdk4/6 Rb kinase activities in the presence of FcγR-derived signals might be explained by a failure to form stable complexes with the regulatory D-type cyclin. To evaluate this, nondenaturing detergent lysates were prepared from B cells and incubated with either anti-Cdk4 or anti-Cdk6 Abs. The resulting immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with anti-cyclin D2 or anti-cyclin D3 Abs. As a positive control for these experiments, we found that both cyclins D2 and D3 coprecipitated with Cdk4 and Cdk6 from lysates of F(ab′)₂ anti-Ig-stimulated B cells (Fig. 6⇓A). In control experiments for the specificity of the immunoprecipitating Abs, nonimmune rabbit IgG bound to protein A-agarose did not coprecipitate cyclins D2 or D3 (Fig. 6⇓A). Most significantly, cyclins D2 and D3 proteins did not coprecipitate with anti-Cdk4 or anti-Cdk6 immune complexes recovered from B cells treated with whole anti-Ig at the time points examined. We interpret these data to mean that D-type cyclins and Cdk4/6 are not expressed as stable holoenzyme complexes in response to BCR-FcγR co-cross-linking.

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FIGURE 6.

Cdk4 and Cdk6 immune complexes isolated from BCR-FcγR co-cross-linked B cells are devoid of detectable D-type cyclins. B cells were cultured in medium alone (lane M), stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig) or whole anti-Ig (wIg) for the indicated time in hours. A, Cell lysates were prepared and immunoprecipitated with 4 μg/ml control rabbit sera (NRS), anti-Cdk4 Ab, or anti-Cdk6 Ab. The immune complexes were then separated by SDS-PAGE and immunoblotted with anti-cyclin D2 and anti-cyclin D3 mAbs (1:500 dilution of hybridoma supernatants). B, Cell lysates were immunoprecipitated with 4 μg/ml anti-cyclin E and anti-cyclin A Abs and the immune complexes immunoblotted with 1.5 μg/ml anti-Cdk2 Ab. Lane J represents immunoblotting of 10 μg activated Jurkat T cell extracts which served as a positive control for the expression of Cdk2.

Protein A-agarose bound anti-cyclin E Ab recovered from whole anti-Ig treated B cells extracts was devoid of detectable Cdk2 at the time points examined (Fig. 6⇑B). These observations are consistent with the lack of detectable Cdk2 protein expression in whole anti-Ig-treated B cells. As a control for the immunoprecipitations, Cdk2 protein coprecipitated with cyclin E immune complexes recovered from parallel BCR-stimulated B lymphocytes at 24 h. The timing of cyclin E/Cdk2 complex assembly in BCR-stimulated B cells is consistent with its role in late G₁ phase of the cell cycle (34, 36). We also monitored cyclin A/Cdk2 complex assembly in BCR-stimulated B cells, which was expressed at a later time in comparison to cyclin E/Cdk2 complexes (Fig. 6⇑B). In mammalian cells, cyclin A/Cdk2 complexes assemble during S phase of the cell cycle, and therefore their assembly serves as an endogenous readout for BCR-stimulated S phase entry.

p27 displays differential expression in B cells stimulated with F(ab′)₂ anti-Ig and whole anti-Ig

We also investigated whether the ability of whole anti-Ig to block G₁-to-S progression was associated with changes in the expression of several known Cdk inhibitors, including Cip1 (p21) and Kip1 (p27) (42). As shown in Fig. 7⇓, p21 levels in F(ab′)₂ anti-Ig-stimulated B cells exhibited a modest increase at 31 h. Expression of p21 in whole anti-Ig-treated B cells was similar to that of F(ab′)₂ anti-Ig-stimulated B cells. Interestingly, the level of p27 in control B cells was relatively high; B cells stimulated with F(ab′)₂ anti-Ig exhibited a decrease in p27 levels at 12 h that was ∼50% of control B cells. The level of p27 declined further at each subsequent time point to a level that was ∼25% of control B cells at 31 h. These results are consistent with studies by Snow and coworkers (10) and others (47), demonstrating decreased p27 levels in response to mitogenic F(ab′)₂ anti-Ig, LPS and CD40 stimulation. Importantly, the BCR-induced decline in p27 levels was blocked in response to co-cross-linking with FcγR.

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FIGURE 7.

Expression of Cip and Kip proteins in BCR-stimulated and BCR-FcγR growth-arrested B cells. B lymphocytes were cultured in medium alone (lane M) or stimulated with 10 μg/ml F(ab′)₂ anti-Ig (Ig) or whole anti-Ig (wIg) for the indicated time in hours. Cell lysates were prepared and 5 μg of protein was separated by SDS-PAGE and immunoblotted with 1.5 μg/ml anti-p21 or anti-p27 Abs as described in Materials and Methods. The m.w. of standard proteins (kDa) is indicated on the left. The p27 autoradiogram was analyzed by densitometry, and the levels of p27 protein represented as arbitrary OD units.