FBXO10 Targets HGAL for Degradation

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 a better prognosis. We previously showed that HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for successful completion of the GC reaction. In our previous studies we demonstrated that upon BCR activation HGAL binds to and increases Syk kinase activity, resulting in enhanced BCR signaling. Syk induces HGAL phosphorylation, leading to its redistribution from lipid rafts to the bulk membrane and cytoplasm, followed by its rapid degradation. The mechanism of HGAL degradation is currently unknown.

To elucidate the mechanism of HGAL degradation, we hypothesized that HGAL is degraded by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. Consequently, we examined the ability of HGAL to be recruited to the SCF by screening a panel of F-box proteins transfected into HEK293T cells by reciprocal coimmunoprecipitations (Co-IPs). These studies revealed that HGAL specifically bound only FBXO10. Concordantly, HGAL Co-IPed with endogenous SKP1 and CUL1 in the presence of FBXO10. Furthermore, a dose dependent reduction of HGAL levels was observed in Raji cells transfected with increasing quantities of FBXO10 expressing plasmid. In contrast, none of the other FBXO11 tested F-box proteins affected HGAL levels. The reduction in HGAL protein level by FBXO10 was due to enhanced proteolysis, as demonstrated by the decrease in HGAL protein half-life and the rescue of its levels by either addition of proteasome inhibitor MG132 or expression of a dominant-negative CUL1 mutant (CUL1 (1-242)). Furthermore, depletion of FBXO10 by two short hairpin RNA (shRNA) constructs in Raji cells increased HGAL stability and half-life.

SCF complexes mediates protein degradation by polyubiquitination. We therefore examined if HGAL protein can be ubiquinated. To this end we cotransfected 293T cells with HGAL-V5, FBXO10-FLAG, HA-ubiquitin with or without CUL1 (1-242) and blotted immunoprecipitated HGAL for ubiquitin. FBXO10 expression markedly increased HGAL ubiquitination and this was significantly decreased in cells coexpressing CUL1 (1-242). Similarly, HGAL ubiquitination was markedly decreased in cells expressing FBXO10 (FBXO10- ΔF-box), in which the F-box motif, necessary for linking the target protein to the SCF ubiquitin ligase moieties, is deleted. These results support the function of FBXO10 in binding HGAL and facilitating its ubiquitination by the SCF complex prior to its degradation.

We next analyzed the functional significance of HGAL degradation by FBXO10 on BCR activation, as measured by intracellular and transmembrane Ca²⁺ mobilization. Overexpression of FBXO10 in Raji cells expressing endogenous HGAL led to decreased levels of HGAL and in Ca²⁺ mobilization. In contrast, FBXO10 did not affect BCR-induced Ca²⁺ mobilization in Raji cells in which HGAL was deleted by crispr/Cas9 targeting. These results suggest that FBXO10 affects BCR signaling via regulating HGAL levels.

While FBXO10 mediates HGAL degradation post BCR-induced HGAL phosphorylation, FBXO10 also bound HGAL in the absence of BCR activation. The binding of FBXO10 to HGAL was unaffected in Raji cells incubated with anti-IgM antibodies. Treatment of cellular lysates with l-phosphatase did not affect FBXO10 binding to HGAL. These findings indicate that FBXO10-dependent degradation of HGAL is phosphorylation independent. In contrast, HGAL degradation by FBXO10 was dependent on HGAL lipid raft localization, since HGAL myristoylation and palmitoylation mutants (G2A, C43A/C45A, and G2A/C43A/C45A) exhibited decreased Co-IP with the FBXO10 protein. To further map FBXO10 binding motifs of HGAL, we generated HGAL deletion and mutant constructs and examined their ability to Co-IP with FBXO10. These studies revealed that HGAL amino acids 91-95(HRVLC) mediate HGAL binding to FBXO10.

In summary, our results demonstrate that SCF^FBXO10 is the ubiquitin ligase for HGAL and regulates BCR signaling via controlling HGAL expression. Some diffuse large cell lymphomas expressing HGAL harbor inactivating mutations or express low levels of FBXO10, thus predisposing to enhanced BCR signaling- a phenomenon implicated in lymphomagenesis.