Integrated data mining characterizes HASPIN as a potential splicing regulatory kinase. (A) Schematic depicting integration of 2 proteomics data sets (Maiolica et al³⁵ and Johnson et al³⁶) to assign HASPIN specificity scores to residues differentially phosphorylated on semispecific HASPIN inhibition. (B) Histogram of HASPIN percentile scores for 3845 differential phosphosites reported by Maiolica et al³⁵ that mapped to the serine/threonine kinase substrate atlas by Johnson et al³⁶. Y-axis is the number of phosphosites with HASPIN percentile scores within indicated histogram bucket range. X-axis is histogram bucket ranges corresponding to substrate atlas HASPIN percentile scores. Colored brackets indicate top deciles of HASPIN substrates. (C) Heat map depicting gene ontology (GO) enrichment for proteins belonging to 70th, 80th, and 90th HASPIN substrate percentiles correspond to bracketed bins in panel B. GO terms and percentiles were grouped by hierarchical clustering as indicated by column and row dendrograms. Cell color represents –log₁₀(P value) for corresponding GO term enrichment. (D) Western blot analysis of phospho-SR, total SR, and α-tubulin (loading control) in Kasumi-1 cells expressing either nontargeting control or 1 of 2 unique HASPIN-targeting shRNAs. SRSF6, SRSF5, and SRSF2 are labeled according to size. Bar plot quantifies the ratio of phosphorylated SR protein signal to total SR protein signal where both values are normalized to respective loading controls and presented as values relative to the control condition. Representative blot of 3 independent experiments revealed. Data are mean ± SD of 3 independent experiments. Significance determined by 2-way ANOVA with Holm-Sidak correction for multiple comparison testing. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (E) Bar plot depicting the fraction of RNA splicing-related HASPIN substrates that are also predicted substrates for indicated splicing regulatory kinases. A total of 229 phosphosites corresponding to RNA splicing proteins enriched in panel C were analyzed. Substrates with >90% kinase percentile score for both HASPIN and indicated kinase were considered mutual or shared targets. Colored and gray areas represent percent of shared or exclusive substrates, respectively. (F) Venn diagram depicting overlap of proteins belonging to experimentally validated HASPIN-interactor data (Maiolica et al³⁵ and BIOGRID) and inferred 90th percentile HASPIN kinase substrates. (G) STRING-DB protein-protein interaction network of experimentally validated HASPIN interacting proteins and inferred 90th percentile HASPIN kinase substrates. Nodes are proteins. Edges are STRING relationships at the highest confidence level (STRING confidence score >0.900). Node color indicates data origin. Inferred 90th percentile HASPIN substrate proteins are colored blue. HASPIN interacting proteins from affinity mass spectrometry data as reported by Maiolica et al³⁵ are colored red. Curated HASPIN interacting proteins from the BIOGRID database are colored green. Colored ellipses represent prominent GO term enriched in nodes contained within. RNA splicing enrichments are colored in orange. Transcription enrichments are colored in purple. Cell cycle enrichments are colored in yellow. A total of 84 singleton nodes are hidden.