Abstract
The chromosome 21 encoded protein kinase DYRK1A is essential for normal human development. Mutations in DYRK1A underlie a spectrum of human developmental disorders, and increased dosage in trisomy 21 is implicated in Down syndrome related pathologies. DYRK1A regulates a diverse array of cellular processes through physical interactions with substrates and binding partners in various subcellular compartments. Despite recent large-scale protein-protein interaction profiling efforts, DYRK1A interactions specific to different subcellular compartments remain largely unknown, impeding progress toward understanding emerging roles for this kinase. Here, we used immunoaffinity purification and quantitative mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. This interactome was enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage, and found that DYRK1A expression and kinase activity are required for maintenance of 53BP1 expression and subsequent recruitment to DNA damage loci. Further, DYRK1A knock out conferred resistance to ionizing radiation in colony formation assays, suggesting that DYRK1A expression decreases cell survival efficiency in response to DNA damage and points to a tumor suppressive role for this kinase.
Highlights
Most of the de novo DYRK1A mutations associated with human neurodevelopmental phenotypes have been shown to disrupt kinase activity in vitro[13,14], a number of clinically relevant non-synonymous mutations outside of the kinase domain failed to disrupt wild-type activity, pointing to kinase-activity independent functions of DYRK1A during brain development[14]
Our results reveal a new role for DYRK1A in DNA damage repair, with potential implications for radioresistance and tumor suppressive mechanisms in cancer
This strategy ensures maximal coverage of interaction partners in the event that an antigenic surface overlaps with a protein interaction interface and disrupts capture of endogenous prey interactions
Summary
Most of the de novo DYRK1A mutations associated with human neurodevelopmental phenotypes have been shown to disrupt kinase activity in vitro[13,14], a number of clinically relevant non-synonymous mutations outside of the kinase domain failed to disrupt wild-type activity, pointing to kinase-activity independent functions of DYRK1A during brain development[14]. Kinase activity-independent roles have been reported for DYRK1A in regulating Arip[4] transcriptional activation[20], and recruitment to serum-responsive promoter elements[21], suggesting that its functions extend beyond phosphorylation to non-catalytic mechanisms such as scaffolding and protein-DNA interactions, as observed for other protein kinases[22]. Non-physiological overexpression of DYRK1A has been shown to alter its subcellular distribution[35], confounding the interpretation of DYRK1A interaction studies that employ ectopic expression. To circumvent these issues and identify DYRK1A protein interactions within the nucleus, we performed mass spectrometry analysis of immunoaffinity-purified endogenous DYRK1A from HeLa nuclear extracts. Our results reveal a new role for DYRK1A in DNA damage repair, with potential implications for radioresistance and tumor suppressive mechanisms in cancer
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