A comprehensive proteomics-based interaction screen that links DYRK1A to RNF169 and to the DNA damage response

Abstract

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have been described, its upstream regulators and downstream targets are still poorly understood, an information that could shed light on the functions of DYRK1A in the cell. Here, we carried out a proteomic screen using antibody-based affinity purification coupled to mass spectrometry to identify proteins that directly or indirectly bind to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed in order to discriminate between true positives and non-specific interactions. Most of the proteins identified in the screen are novel candidate DYRK1A interactors linked to a variety of activities in the cell. The in-depth characterization of DYRK1A’s functional interaction with one of them, the E3 ubiquitin ligase RNF169, revealed a role for this kinase in the DNA damage response. We found that RNF169 is a DYRK1A substrate and we identified several of its phosphorylation sites. In particular, one of these sites appears to modify the ability of RNF169 to displace 53BP1 from sites of DNA damage. Indeed, DYRK1A depletion increases cell sensitivity to ionizing irradiation. Therefore, our unbiased proteomic screen has revealed a novel activity of DYRK1A, expanding the complex role of this kinase in controlling cell homeostasis.

DYRK1A Overexpression Alters Cognition and Neural-Related Proteomic Pathways in the Hippocampus That Are Rescued by Green Tea Extract and/or Environmental Enrichment

“…DYRK1A interactors were taken from the mammalian verified interactors reported in previous literature (Aranda et al, 2011; Duchon and Herault, 2016; Guard et al, 2019; Roewenstrunk et al, 2019). The significance of the overlaps between DYRK1A interactors and the differentially abundant proteins, or between DYRK1A interactors and the proteins with differentially abundant phosphopeptides, was assessed using a Fisher exact test.…”

The DYRK Family of Kinases in Cancer: Molecular Functions and Therapeutic Opportunities

“…All human DYRKs accumulate in the cytosol of cells, and DYRK1A, DYRK2 and DYRK4 can be imported into the nucleus by means of dedicated NLSs [ 7 , 8 , 9 ]. DYRK1A translocation to the nucleus acquires special biological significance, since it has been described as a chromatin-associated kinase capable of regulating the gene expression [ 10 , 11 ], and it is functionally linked to the DNA damage response (DDR) [ 12 , 13 , 14 ]. Chromatin association in the DDR context has also been recently described for DYRK1B [ 15 ].…”

Differential DNA Methylation in Prostate Tumors from Puerto Rican Men

“…RNF169 is recognized as a substrate for DYRK1A . RNF169 phosphorylation at S688 plays a major role in removing 53BP1 from the DNA damage foci [ 52 ]. Furthermore, RNF169 interacts with DYRK1A and localizes to DNA damage foci by binding [ 53 ].…”