GCET2, a New Adaptor Protein in B Cell Signaling.

GCET2 (Germinal Center B-cell Expressed Transcript 2; also named HGAL, Human Germinal center-Associated Lymphoma) is a newly cloned gene that has been shown to be a useful marker for germinal center (GC) B-cells and GC B-cell derived malignancies, including follicular lymphomas and germinal center B cell-like diffuse large B-cell lymphomas (GCB-DLBCLs). GCET2 is also a prognostic indicator for DLBCLs, and patients with higher expression of GCET2 have significantly better survival than those with lower expression. We cloned GCET2 from a number of GC B cell-restricted expressed sequence tags (ESTs) in order to study the differentiation of GC B-cells and to elucidate the mechanisms underlying the GC reaction, which is not well understood. We are report here the biochemical and biological properties of GCET2, which may help to determine its role in the GC reaction. Sequence analysis of GCET2 did not reveal any known domains but predicted five tyrosine phosphorylation sites, all of which are conserved in its mouse homologue, M17, also a GC B cell-restricted transcript. We first determined the cellular localization of GCET2 using subcellular fractionation of a B cell line, DHL16, and found that GCET2 is constitutively localized in cellular membranes but is excluded from lipid rafts. These findings were further confirmed by fluorescence confocal microscopy. GCET2 does not have a transmembrane domain but has a putative myristoylation site and a putative palmitoylation site, which may mediate its membrane attachment. Using ³H metabolic labeling, we demonstrated that GCET2 was both myristoylated and palmitoylated, and GCET2 lost its membrane association after mutating both of these sites, indicating that the membrane attachment of GCET2 is mediated by these two post-translational modifications. We then studied tyrosine phosphorylation of GCET2. In both Daudi and DHL16 cells, GCET2 was phosphorylated following pervanadate treatment. By serially mutating the five predicated tyrosine-phosphorylation sites, we found that the distal three sites are crucial for GCET2 phosphorylation. GCET2 was also phosphorylated when co-transfected into COS7 cells with protein tyrosine kinases (PTKs) LYN, LCK or SYK, and therefore it may be a substrate of these kinases in B cells. GCET2 has a conserved GRB2 binding site, and it indeed associates with GRB2 following pervanadate treatment. Our data suggest that GCET2 acts as an adaptor protein in GC B-cells by transducing signals from GC B-cell membrane to the cytosol. Our working model is as follows: a stimulus to GC B-cells induces the activation of PTKs LYN and SYK, which sequentially phosphorylate GCET2 at the plasma membrane. Phosphorylated GCET2 then recruits GRB2 from the cytosol to the plasma membrane, and this complex further recruits additional partners and activates downstream pathways, which function in the GC reaction. We are currently identifying other proteins in the GCET2/GRB2 complex to determine the pathways downstream of GCET2 activation.