Interaction of HGAL with Grb2 Enhances RhoA and B-Cell Receptor Signaling

Abstract 681

Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by improved survival. Previously we have demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by 1) interaction with F-actin and myosin II, inhibiting the ability of myosin to translocate actin; 2) activating RhoA signaling by direct interactions with RhoA-specific guanine nucleotide exchange factors. We recently demonstrated that HGAL enhances B-cell receptor (BCR)-mediated Syk activation by directly binding to and enhancing Syk kinase activity. However, the precise molecular mechanisms of the HGAL effects on these signaling pathways and requirement for cofactors have yet to be described. Examination of the HGAL protein sequence revealed the presence of a putative growth factor receptor-bound protein 2 (Grb2) binding motif (YYENV; aa106-110). Grb2 is an abundant adaptor protein with important functions in multiple intracellular pathways, including BCR and RhoA signaling. We examined whether Grb2 may directly interact with the HGAL protein and be involved in regulation of HGAL-mediated signaling. Confocal microscopy studies demonstrated colocalization of HGAL and Grb2 proteins in Raji and HeLa cell lines, the latter transiently expressing HGAL protein. Concordantly, coimmunoprecipitation experiments in VAL and Raji lymphoma cell lines detected endogenous Grb2 in immunoprecipitates of endogenous HGAL and vice versa. GST pull down assay using recombinant GST-Grb2 failed to pull down non-phosphorylated TRX-HGAL protein. In contrast, TRX-HGAL protein in vitro phosphorylated by Lyn kinase was detected in the GST-Grb2 pull down, confirming a direct interaction between these proteins and demonstrating requirement for HGAL phosphorylation, as would be predicted from the YYENV binding motif. We further elucidated the relevance of this interaction by evaluating the effects of siRNA mediated knockdown of Grb2, HGAL or both proteins together on BCR and RhoA signaling in lymphoma cell lines. In accordance with our previous data, HGAL knockdown led to a significant decrease in phosphorylated Syk (Y352) following BCR stimulation. A similar decrease in Syk(P352) phosphorylation was observed following knockdown of Grb2 alone or simultaneously with HGAL. Concordantly, BCR-induced Ca²⁺ mobilization was decreased significantly and to a similar extent following HGAL or Grb2 knockdown. Concomitant knockdown of HGAL and Grb2 did not lead to additive or synergistic decrease in BCR-induced Ca²⁺ mobilization. Similarly, knockdown of HGAL or Grb2 decreased fibronectin(FN)-induced RhoA-GTP levels. To further elucidate the importance of the HGAL-Grb2 interaction, we have generated a plasmid encoding a HGAL(Y106FY107F) protein in which the putative Grb2 binding site was mutated. The HGAL(Y106FY107F) protein is not interacting with the Grb2 protein, as demonstrated by coimmunoprecipitation studies. Expression of the wild type HGAL protein in HeLa cells markedly increased both FN-induced RhoA-GTP levels and RhoA-mediated serum response factor (SRF)-induced transcriptional activity from the serum responsive element (SRE). In contrast, the HGAL(Y106FY107F) mutant did not affect either FN-induced RhoA-GTP levels or SRF-induced transcriptional activity from the SRE. These observations suggest that the HGAL-Grb2 interaction is important for mediating HGAL's effect on RhoA signaling pathway. HGAL was previously shown to inhibit lymphoma cell motility by activating the RhoA signaling pathway. Indeed, siRNA mediated HGAL knockdown significantly increased lymphoma cell chemotaxis in response to IL-6 and SDF1. Knockdown of Grb2 similarly increased IL-6-induced lymphoma chemotaxis, with no further increases in IL-6 induced chemotaxis observed flowing concomitant HGAL and Grb2 knockdown. In contrast, compared to the HGAL knockdown, knockdown of Grb2 significantly increased lymphoma cell chemotaxis in response to SDF-1. Concomitant knockdown of both Grb2 and HGAL resulted in an ever greater increase in lymphoma cell motility in response to SDF-1. In summary, our findings suggest that interaction of HGAL with Grb2 protein enhances both RhoA and B-cell receptor signaling and may be necessary for mediating at least some of the HGAL effects.