Stimulation of the B-cell or T-cell receptor triggers assembly of an intracellular complex composed of three proteins, CARMA1, BCL10, and MALT1 (CBM), which then induces activation of the NF-κB transcription factor and lymphocyte proliferation. Dysregulated CBM signaling, which occurs as a result of several gain-of-function mutations, underlies a variety of lymphoid malignancies including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL). Multiple studies established that inhibiting MALT1 reduces growth and viability of ABC-DLBCL. We recently identified G-protein-coupled receptor kinase 2 (GRK2), one of seven members of the GRK family, as a novel MALT1-binding protein and demonstrated that GRK2 inhibits MALT1-dependent NF-κB activation. Further, knockdown of GRK2 in ABC-DLBCL enhances tumor growth, suggesting that GRK2 may act as a tumor suppressor by binding and inhibiting MALT1. As a first step toward elucidating how GRK2 regulates MALT1, we sought to identify the specific site(s) within GRK2 that interacts with MALT1. We created a series of expression constructs encoding specific GRK2 fragments and tested the ability of these fragments to inhibit CBM-dependent NF-κB activation. “GRK2 N-term” (AA1-173) includes the N-terminal helix (αN) and the regulator of G-protein signaling homology (RH) domain while “GRK2 ΔαN” (AA 30-C) lacks αN and retains all other domains. Using an NF-κB reporter assay, we found that GRK2 N-term and GRK2 ΔαN each block CBM-dependent NF-κB activation. These results suggest that RH, the only domain retained in both GRK2 fragments, is sufficient to inhibit MALT1. Since the GRK2 RH domain is known to interact with the alpha subunit of guanine-nucleotide binding proteins (Gα), we hypothesized that this protein interaction site may also mediate GRK2 RH binding to MALT1. However, we found that mutations that abrogate GRK2 binding to Gα do not affect GRK2's ability to inhibit CBM-dependent NF-κB activation, suggesting that GRK2 interacts with MALT1 via a different site. We next compared the ability of GRK family members, which each have structurally homologous RH domains, to inhibit CBM-dependent NF-κB activity. We found that, like GRK2, GRK3 inhibits CBM-dependent NF-κB activity while GRK1 does not. We are now comparing RH domain structures for GRK2, GRK3, and GRK1 to identify structural requirements for RH interaction with MALT1. Also, we performed in silico modeling, which predicts that a short stretch of amino acids within GRK2 RH, designated “P1-P2,” interacts with the MALT1 Death Domain. Together, our data suggest that the GRK2 RH domain is sufficient to inhibit MALT1. Ongoing work aims to narrow down the region(s) within the RH that mediates GRK2 binding and inhibition of MALT1. We hope to utilize these analyses to inform the development of a novel class of MALT1 inhibitors for treatment of MALT1-dependent leukemia and lymphoma.
Citation Format: Lisa M. Maurer, Matthew Trotta, Jing Cheng, Beibei Bill Chen, Peter C. Lucas, Linda M. McAllister-Lucas. Structural requirements for GRK2-mediated inhibition of the MALT1 proto-oncoprotein [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-13.