Mapping the SRPK1 phosphorylation sites in the HBc CTD by mutation and MS.

(A) Position of S/T>A mutations, dominant m/z peak observed and calculated MH+ mass of best-fitting phospho-species. Note that S181A is the only single-site variant with a best-fit m/z for seven-fold phosphorylation; all other single-site mutants showed the best match to six phosphoryl groups. (B) Impact of number and position of phospho-sites on Phos-tag SDS-PAGE mobility. Following Phos-tag SDS-PAGE HBc proteins were analyzed by immunoblotting using the HBc assembly-domain specific mAb 1D8. All SRPK1-coexpressed proteins contained seven (HBc183, S181A) or six phosphoryl groups (all others) but variants S170A (*) and especially T160A and S162A (**) were less retarded, even though much more than three-fold PKAcd phosphorylated HB183. The impact of the mutations on recognition by the phospho-CTD specific mAb T2212 is shown in S12 Fig. (C) Impact on CLP RNA content. CLPs from the indicated HBc proteins were analyzed by NAGE and sequential SG2 vs. SR staining using an improved, background-reducing protocol (Materials and Methods). SG2: SR ratios are indicated in percent of that in non-phosphorylated HBc183 CLPs. Bars show the mean of ≥3 determinations; error bars represent standard deviation (SD). Original NAGE fluorograms plus data for additional variants are shown in S8 Fig. Note the exceptionally low SG2 to SR ratio for S181A and the higher ratio for S170A vs. all other single S/T>A variants (red arrows). The minor impact of PKA and PKC coexpression on RNA content (blue bars) correlated with the predominance of only three-fold phosphorylated species (S7 Fig) and their less pronounced retardation in Phos-tag SDS-PAGE (Fig 5B and Fig 9C).