GRK2 Is a Novel Modulator of the MALT1 Oncoprotein

Abstract

Background and Significance: Lymphocyte antigen receptor-dependent stimulation of the NF-κB transcription factor is a key event during the normal adaptive immune response. However, dysregulated activation of these signaling pathways can lead to lymphoma. Stimulation of the B-or T-cell receptor promotes assembly of the CARMA1-Bcl10-MALT1 (CBM) signaling complex, where MALT1 functions as the essential effector molecule by carrying out two important functions: First, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, acts as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of canonical NF-κB signaling. Activating mutations in either BCR or CARMA1 promote constitutive and exaggerated MALT1 activity and are seen as driver mutations in diffuse large B cell lymphoma (DLBCL). Aberrant MALT1 activity also underlies the pathogenesis of MALT lymphoma, another subtype of B-cell malignancy. As such, MALT1 has emerged as a potential new target for pharmaceutical inhibition in the treatment of lymphoma. Here, we describe our discovery of a novel MALT1-binding partner, G protein-coupled receptor kinase 2 (GRK2). We hypothesize that GRK2 plays a critical role in the pathogenesis of lymphoma by modulating MALT1 activity.Methods and Results: Using co-immunoprecipitation and liquid chromatography mass spectrometry we identified GRK2 as a novel MALT1-interacting protein. We validated our results by showing that endogenous GRK2 and MALT1 co-immunoprecipitate (CO-IP) in both B cells and T cells. We next utilized GST-tagged purified proteins to demonstrate a direct interaction between GRK2 and MALT1. We then performed a number of studies to investigate the functional consequences of GRK2 interaction with MALT1. A CO-IP assay demonstrated that GRK2 inhibits Bcl10-dependent binding to MALT1, and a luciferase reporter assay revealed that GRK2 inhibits Bcl10/MALT1-dependent NF-κB activity. In addition, we demonstrated that GRK2 also inhibits MALT1 proteolytic activity using Western blot analysis of two known MALT1 substrates, the deubiquitinase cylindromatosis (CYLD) and the NF-kB family member RelB. Moreover, in Jurkat T cells, overexpression of GRK2 impeded AgR-induced NF-κB activation while stable GRK2 knockdown resulted in enhanced AgR-induced phosphorylation of inhibitor of kappaB (IκB) and secretion of interleukin2 (IL-2).We next began to investigate the potential of GRK2 to function as a tumor suppressor by blocking MALT1 activity. Of note, utilizing a published data set to compare GRK2 mRNA expression in 44 DLBCL patient samples to that of 20 samples of healthy B-cell controls, we found that GRK2 mRNA levels are markedly lower in DLBCL cases. We also compared GRK2 mRNA levels among patients diagnosed with DLBCL grouped by vital status, using data from the Cancer Genome Atlas (TCGA). We found that tumors from patients who died of disease within three years expressed significantly lower levels of GRK2 as compared to tumors from patients alive after three years. We also wished to investigate the impact of GRK2 expression on activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) cells, which require MALT1 activity for growth and survival. We were successful in engineering GRK2 overexpression in control germinal center B-cell like (GCB) DLBCL cells, which do not depend on MALT1 activity for growth. In contrast, we found that in ABC-DLBCL cells, which do depend on MALT1 activity, GRK2 overexpression greatly impairs cell proliferation, making it difficult to grow these engineered cells. These data provide support for the concept that GRK2 may have a potential tumor suppressor role in MALT1-dependent lymphoma.Conclusions: Our study represents the first report of a negative modulator of MALT1 and provides critical new insight into mechanisms regulating its activity. We hope to harness this information in developing novel strategies for inhibiting MALT1-dependent lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.