Rapid STAT phosphorylation via the B cell receptor. Modulatory role of CD19

Abstract

Engagement of the B cell receptor (BCR) initiates multiple signaling cascades which mediate different biological responses, depending on the stage of B cell differentiation, antigen binding affinity, and duration of stimulation. Aggregation of co-receptors such as CD19 with the antigen receptor has been suggested to modulate the signals necessary for the development and functioning of the humoral immune system. In this study, we demonstrate that engagement of the antigen receptor on peripheral blood B cells, but not naïve splenic B lymphocytes, leads to rapid phosphorylation of signal transducers and activators of transcription 1 (STAT1) on Tyr-701 and Ser-727. Interestingly, phosphorylation on tyrosine diminished with increased stimulation, whereas serine phosphorylation correlated directly with the level of BCR cross-linking. In contrast, phosphorylation of STAT3 occurs exclusively on serine and is sensitive to inhibitors of the PI3-kinase and the ERK1/2 pathways. Finally, we show that co-ligation of CD19 with the BCR results in increased tyrosine phosphorylation of STAT1 relative to BCR cross-linking alone, establishing CD19 as a positive modulator of BCR-mediated STAT activation.

Fig. 1. Rapid STAT1 phosphorylation via the antigen receptor

A, induction of STAT1 Tyr-701 phosphorylation. RAMOS cells were left untreated (lane 1), treated with 1000 units/ml IFNα (lane 2), or 1 μg/ml anti-IgM antibody (lanes 3–9) for the indicated times. Proteins were probed with phospho-(Y701)-specific STAT1 antibody (upper panel) and reprobed with anti-STAT1 antibody to verify equal protein amounts (lower panel). B, decrease of STAT1 tyrosine phosphorylation at high levels of BCR cross-linking. RAMOS cells treated with increasing amounts of anti-IgM antibody at 2′ and 30′, and lysates were probed with phospho-(Y701)-specific STAT1 antibody (upper panel). The blot was reprobed with anti-STAT1 antibody to verify equal protein amounts (lower panel). Both blots were quantitated by densitometry, and STAT1 phosphotyrosine content normalized for STAT1 levels is displayed below. C, STAT1 Ser-727 phosphorylation follows a strict dose-response correlation. RAMOS cells were treated with the concentration of anti-IgM antibody for 30′, and the resolved proteins were probed with phospho-(S727)-specific STAT1 antibody (upper panel). The blot was reprobed with anti-STAT1 antibody to verify equal protein amounts (lower panel). D, lack of STAT1 Tyr-701 phosphorylation via the BCR in murine B cells. Primary murine splenocytes were stimulated with 1000 units/ml muIFNα (lane 2) or with the indicated concentrations of anti-(mu) IgM antibodies (lanes 3–7) for 2 min, and lysates were probed with phospho-(Y701)-specific STAT1 antibody (upper panel). The lower part of the blot was probed with anti-phospho-specific ERK1/2 antibody to verify effectiveness of BCR stimulations (lower panel). E, rapid STAT1 Tyr-701 phosphorylation in human and murine PBLs. Human or murine peripheral blood B lymphocytes were stimulated with the indicated concentrations of anti-IgM antibodies for 5 min, and lysates were probed with phospho-(Y701)-specific STAT1 antibody (upper panel). The lower part of the blot was probed with STAT1 antibody to verify equal protein amounts (lower panel).

Fig. 2. Selective STAT3 phosphorylation on Ser-727

A, lack of STAT3 Tyr-705 phosphorylation. RAMOS cells were left untreated (lane 1), treated with 1000 units/ml IFNα (lane 2), or 1 μg/ml anti-IgM antibody (lanes 3–9) for indicated times. The proteins were probed with phospho-(Y705)-specific STAT3 antibody (upper panel) and reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel). B, rapid STAT3 Ser-727 phosphorylation. Extracts shown in panel A were resolved by SDS-PAGE and probed with phospho-(S727)-specific STAT3 antibody. The blot was reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel). C, p44/42 MAP kinase activation via the BCR. Extracts shown in panel A were resolved by SDS-PAGE and probed with phospho-ERK1/2 specific antibody. The blot was reprobed with anti-ERK2 antibody to verify equal protein amounts (lower panel). D, lack of STAT3 tyrosine phosphorylation is concentration-independent. RAMOS cells were left untreated (lane 1), or treated with 1000 units/ml IFNα (lane 2), or increasing amounts of anti-IgM antibody for 30′, and lysates were probed with phospho(Y705)-specific STAT3 antibody. The blot was reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel). E, STAT3 serine 727 phosphorylation correlates with the intensity of stimulation. Extracts shown in panel D were resolved by SDS-PAGE and probed with phospho-(S727)-specific STAT3 antibody. The blot was reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel).

Fig. 3. STAT3 serine phosphorylation requires ERK1/2 and PI3-kinase activity

A, inhibition of BCR-mediated MAP-kinase activation. RAMOS cells were pretreated with 50 nM wortmannin (lane 3), or 20 (PD_L) or 100 μM (PD_H) PD98059 (lanes 4 and 5) for 60′ prior to stimulation with 1 μg/ml anti-IgM antibody for the indicated time. ERK1/2 activation was assessed by probing with phospho-ERK1/2 specific antibody (upper panel). The blot was reprobed with anti-ERK2 antibody to verify equal protein amounts (lower panel). B, inhibition of ERK1/2 activation abrogates STAT3 serine phosphorylation. Extracts shown in panel A were resolved by SDS-PAGE and probed with phospho-(S727)-specific STAT3 antibody (upper panel). The blot was reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel). C, prevention of STAT3 serine phosphorylation does not restore tyrosine phosphorylation. RAMOS cells were pretreated with 50 nM wortmannin (lanes 3 and 7), or 20 μM (PD_L) or 100 μM (PD_H) PD98059 (lanes 4 and 8 and 5 and 9, respectively) for 60′ prior to stimulation with either 1 μg/ml anti-IgM antibody (lanes 2-5) or 1000 units/ml IFNα (lanes 6–9) for the indicated time. The proteins were probed with phospho-(Y705)-specific STAT3 antibody (upper panel). The blot was reprobed with anti-STAT3 antibody to verify equal protein amounts (lower panel).

Fig. 4. CD19 is a positive modulator of STAT1 tyrosine phosphorylation via the antigen receptor

A, co-ligation of CD19 and the BCR enhances MAP kinase activation. RAMOS cells were left untreated (lane 1), or treated with 0.1 μg/ml biotinylated anti-IgM antibody + 10 μg/ml avidin in the absence (lanes 2 and 5), or presence (lanes 3–4 and 6 and 7) of the indicated increasing amounts of biotinylated agonistic CD19 antibody for either 2′ (lanes 2–4) or 30′ (lanes 5–7). ERK1/2 activation was analyzed by probing with phospho-ERK1/2 specific antibody (upper panel). The blot was reprobed with anti-ERK2 antibody to verify equal protein amounts (lower panel). B, co-ligation of CD19 with the BCR augments STAT1 tyrosine phosphorylation. Extracts shown in panel A were resolved by SDS-PAGE and probed with phospho-(Y701)-specific STAT1 antibody (upper panel). The blot was reprobed with anti-STAT1 antibody to verify equal protein amounts (lower panel).