(A) Schematic of dTAG-VRK1 degrader system. The conjugated FKBP12^F36V binding domain allows small molecule–mediated (dTAG^V-1) recruitment of the VHL ubiquitin ligase complex, targeting exogenous VRK1 for proteasomal degradation. (B) Schematic of VRK1 degrader experiments. Exogenous dTAG-VRK1 is transduced to rescue CRISPR KO of endogenous VRK1. Exogenous dTAG-VRK1 is then under the control of the small molecule degrader (dTAG^V-1) allowing for acute downregulation. (C) Immunoblot validation of the dTAG-VRK1 degrader system in NB-1 neuroblastoma cells. Exogenous dTAG-VRK1 was degraded with dTAG^V-1. Endogenous VRK1 was independently targeted with CRISPR KO. sgLacZ is a nontargeting guide control. (D) Cell viability analysis of dTAG-VRK1-NB-1 cells following addition of either vehicle control or 0.5 μM dTAG^V-1. Significance at each time point was determined by 2-way ANOVA (treatment × time). *P < 0.05, **P < 0.001. (E) Schematic of the quantitative, global phospho-proteomic experiment. Samples were generated in triplicate at 4 hours and 8 hours after dTAG^V-1 (0.5 μM) addition. Following trypsin digestion, peptides were tagged with isobaric tandem mass tags (TMTs), then combined. Phospho-enrichment was performed using IMACs, and then peptides were run on an Orbitrap mass spectrometer. MS2 spectra offer peptide IDs and sample deconvolution through attached mass tags. (F) KSEA of phosphorylation site dynamics following acute degradation of exogenous VRK1. Kinase substrates of CDK1 and AURKA were significantly downregulated following degradation (blue), while substrates of WEE1, BRSK1, and ATM were significantly upregulated (red). (G) Top panel: Venn diagram showing number of unique proteins with a decrease in phosphorylation for at least 1 phosphorylation site in dTAG^V-1–treated samples. Bottom panel: Dot plots showing the overlap of downregulated protein phosphorylation (208 proteins) with select categories of the C5 MSigDB library. All gene sets have FDR ≤ 0.05 as determined by 1-tailed Fisher’s exact test.