Fig 1: In vitro kinase assay validation of hnRNPM phosphorylation by MASTL. (a) The ion abundance of each phospho-site activated in triplicate reactions between MASTL kinase and hnRNPM (as detailed in this figure legend). Phospho-site abundances were calculated using label-free quantitation. Square brackets indicate peptide locations within the protein on which each phospho-site is found. Single hashtags denote a co-modification within that peptide, e.g., carbamidomethylation, deamidation, and/or oxidation. Double hashtags denote a different combination of co-modifications within the same peptide (detailed in Supplementary Table S7). Error bars display the standard error of the mean from three (n = 3) independent reactions. (b) Schematic of hnRNPM showing nuclear localisation domain (NLS) RNA recognition motifs (RRM) and phospho-sites currently annotated in PhosphoSitePlus (Chk1 and PRP4) along with the novel MASTL sites identified here. Sequence alignments of the MASTL phosphorylation sites within hnRNPM (S-633 and S-637), compared to validated substrates Arpp19/ENSA and the possible MASTL motif identified from our SILAC screen (Fig. 2d).
Fig 2: In vitro kinase assay validation of ENSA phosphorylation by MASTL. (a) Venn diagram comparing previously identified MASTL interacting proteins by co-immunoprecipitation55 with those identified from A, and (b) subsequent STRING cluster analysis49 of common proteins. (c) The abundance of each ENSA phospho-site activated in reactions between MASTL kinase and ENSA. The ion abundance for the #Ser67 [64–71] phospho-peptide is normalized to the 60 min control (CTL) reaction. Error bars display the standard error of the mean from three independent reactions. Square brackets indicate peptide locations within the protein on which each phospho-site was found. Hashtags denote a co-modification within that peptide, e.g., carbamidomethylation, deamidation, and/or oxidation (detailed in Supplementary Table S6). (d) Western blot confirmation of ENSA phosphorylation at S-67 in the presence of active MASTL kinase. Total ENSA was used as the loading control and normalization factor for densitometry analysis. P-ENSA/Total was further normalized to the 60 min control (CTL) reaction. Error bars display the standard error of the mean from three independent reactions. (e) The abundance of MASTL phospho-sites present in reactions with ENSA are represented here as a heat map. Two reaction conditions were tested (60 min incubation in presence of 0.5 mM ATP or 1 mM ATP). Phospho-site abundances were calculated using label-free quantitation.
Supplier Page from Abcam for Recombinant Human ENSA protein (His tag N-Terminus)