Abstract 343: Proteomic and functional studies identify DCAF7 as major partner of DYRK1A

Human DYRK1A protein kinase is encoded by dosage-dependent gene since an extra copy contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe developmental defects. Current knowledge of DYRK1A’s role in phosphorylation of proteins involved in cell cycle control, transcription and tumor suppression does not fully explain dosage-dependent function of this important kinase. Using MudPIT proteomic analysis, we identified DYRK1A interacting proteins in human T98G cell line. Four independent DYRK1A-HA pull-down samples were analyzed to identify 50 proteins that were specifically detected in 3 out of 4 replicates. This analysis identified WD-repeat protein DCAF7 as major partner of DYRK1A that was most highly enriched among DYRK1A-binding proteins. Furthermore, glycerol gradient ultracentrifugation showed almost exact co-fractionation of DCAF7 and DYRK1A. DCAF7 has been shown to bind several protein kinases including DYRK1A, DYRK1B, HIPK2 and MAP3K1 suggesting that it serves as a scaffold protein in signal transduction. However, the function of DCAF7 is not established yet. In order to understand the functional relationship between DCAF7 and DYRK1A, we undertook MudPIT proteomic analysis of DCAF7 binding proteins in T98G cells and compared the data with the DYRK1A interactome dataset. Analysis of three independent DCAF7-HA pull-down samples identified 32 proteins specifically detected in all three replicates including DCAF7, DYRK1A and five proteins earlier detected in DYRK1A MudPIT analysis. Given the proposed role of DCAF7 as a scaffold protein, we tested whether DCAF7 mediates its interaction with some of its interacting proteins. Interestingly, we found that RNF169, a recently characterized RING-domain ubiquitin ligase involved in the DNA double-strand break (DSB) repair, binds to N-terminus of DYRK1A independently of DCAF7. Furthermore, our data show that DYRK1A is required for DCAF7-RNF169 interaction because this interaction was abolished in human U-2 OS cells where DYRK1A gene was disrupted using CRISPR-Cas9 editing (DYRK1A-KO cells). Interestingly, stable overexpression of DCAF7 in DYRK1A-KO U-2 OS cells rescued some of the phenotypes observed in these cells, suggesting that DCAF7 could be an effector downstream of DYRK1A. Our findings revealed an unexpected scaffolding role of DYRK1A that is required for mediating the interaction between DNA repair protein RNF169 and the WD-repeat protein DCAF7. While further studies are needed to understand the exact role of the RNF169-DCAF7 interaction in DNA repair, the novel role of DYRK1A as a scaffold protein could explain some aspects of the dosage-dependent function of this protein kinase. In addition, we report here an initial functional characterization of the DCAF7-DYRK1A interaction in human cells and discuss novel functional interactions of DCAF7 that could be independent of DYRK1A. Citation Format: Varsha Anathapadmanabhan, Selene Swanson, Siddharth Saini, Vijay Menon, Larisa Litovchick. Proteomic and functional studies identify DCAF7 as major partner of DYRK1A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2017-343