Grb2 Directly Interacts with HGAL and Ameliorates Its Effects on B-Cell Receptor Signaling

Human Germinal center Associated Lymphoma (HGAL) is specifically expressed in germinal center (GC) B-cells and GC-derived lymphomas. High expression of HGAL is an independent predictor of prolonged survival of Diffuse Large B-Cell (DLBCL) and classical Hodgkin (cHL) lymphoma patients. HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. HGAL increases BCR signaling by binding to and enhancing Syk kinase activity. However, our previous studies also suggested that other proteins may be involved in HGAL-mediated regulation of BCR signaling.

In vitro kinase assays demonstrated that both Syk and Lyn can phosphorylate HGAL. Mass spectrometry (μ LC/MS/MS) demonstrated that these kinases can phosphorylate HGAL's tyrosines Y80, Y86, Y106Y107, Y128 and Y148. The HGAL Y106Y107 comprise a YYENV motif (aa 106-110) similar to the phosphopeptide motif pYXNX frequently used as a binding site to the SH2 domain of Growth Factor Receptor bound protein 2 (Grb2). Grb2 signaling in B cells controls lymphoid follicle organization and the GC reaction. Specifically, Grb2 is an integral component of the BCR signalosome and decreases BCR-induced Ca2+influx. The presence of the phosphorylated YYENV motif in HGAL raised the hypothesis that HGAL-Grb2 interactions may play a role in HGAL -mediated regulation of BCR signaling. To address this possibility, we performed reciprocal coimmunoprecipitations (Co-IPs) of endogenous HGAL and Grb2 in Raji and VAL lymphoma cell lines. These studies demonstrated that HGAL Co-IPs with Grb2. The interaction between these two proteins is dependent on the presence and phosphorylation of tyrosines in the YYENV motif, since an HGAL mutant in which these tyrosines were mutated to phenylalanine (FFENV) failed to Co-IP with Grb2. Isothermal titration calorimetry confirmed that phosphorylated (pYEN) but not unphosphorylated (YEN) HGAL-derived 12-mer peptides bind to the SH2 domain of Grb2 with an affinity of 5µM. GST-Grb2 pull down assays with recombinant Trx-HGAL(FFENV) and Trx-HGAL proteins confirmed that the HGAL-Grb2 interaction is direct and occurs only if the HGAL tyrosines are phosphorylated. Concordantly, addition of phosphatase to cellular lysates decreased the HGAL-Grb2 interaction. Furthermore, CO-IP studies demonstrated that HGAL's interaction with Grb2 increases following BCR stimulation-induced HGAL phosphorylation. Concordantly, confocal microscopy studies demonstrated HGAL-Grb2 colocalization in the cell membrane following BCR signaling activation.

We next examined the functional significance of the HGAL-Grb2 interaction on BCR activation as measured by intracellular and transmembrane Ca2+ mobilization and phosphorylation of proximal BCR effectors (Syk (Y352), BLNK (Y84), BTK (Y551) and PLCγ2 (Y753) in several lymphoma cell lines (U2942, TMD8 and Mino) stablly transfected to express HGAL protein. HGAL expression markedly increased Ca2+ influx and phosphorylation of these proteins, while Grb2 knockdown only slightly increased transmembrane Ca2+ mobilization. Of note, concomitant HGAL expression and Grb2 knockdown further increased intracellular and transmembrane Ca2+ influx and phosphorylation of BCR effectors in comparison to HGAL expression alone. Expression of the HGAL (FFENV) mutant also enhanced Ca2+ influx and phosphorylation of BCR effectors in comparison to wild type HGAL. Concordantly, expression of the dominant negative Grb2 (W193K) mutant also enhanced HGAL's effects on BCR signaling. These observations suggest that Grb2's interaction with HGAL ameliorates HGAL's effects on BCR signaling.

We previously showed that HGAL interacts with Syk and enhances Syk kinase activity. We now demonstrate that Grb2 Co-IPs with both Syk and HGAL and thus may potentially interfere with HGAL-Syk interaction. Indeed, knockdown of Grb2 increased HGAL Co-IP with the Syk kinase and this was associated with increased BCR signaling. These findings indicate that Grb2 ameliorates HGAL-mediated enhancement of BCR signaling by decreasing HGAL binding to Syk.

In summary, out data demonstrates that Grb2 directly interacts with HGAL and ameliorates HGAL-enhanced BCR signaling. These interactions may play an important function in regulating the magnitude of BCR signaling during the GC reaction.