The Drosophila Splicing Factor PSI Is Phosphorylated by Casein Kinase II and Tousled-Like Kinase

<div><p>Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the <i>Drosophila</i> splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in <i>Drosophila</i> S2 tissue culture cells, the majority of which are splicing factors.</p> </div

Biochemical purification of Drosophila casein kinase II.

<p>A) Purification strategy for endogenous casein kinase II. B) Protein composition of peak fraction of activity from the final heparin column visualized by SDS-PAGE and silver-staining. Species identified as casein kinase II alpha and beta are labeled. Bands labeled with an asterisk correspond to contaminating keratin. C) <i>In vitro</i> kinase assay of PSI mutant proteins. Serine to alanine PSI mutant proteins were phosphorylated <i>in vitro</i> using the peak fraction of activity from the final heparin column and using purified recombinant human casein kinase II (NEB P6010S). Assays were visualized using autoradiography, and, to ensure equal protein loading, coomassie staining.</p

Biochemcial fractionation and analysis of the PSI kinase.

<p>A) Purified recombinant PSI and PSI purified from Kc cells was treated with calf intestinal phosphatase (CIP) and then visualized by immunoblotting. B) MS2 spectra identifying phosphopeptides found in PSI. B and Y series ions and neutral loss of phosphate are indicated. Inset: sequence of the phosphopeptide and SEQUEST statistics. MS3 spectra and corresponding spectra of unmodified peptides are given in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056401#pone-0056401-g001" target="_blank">Figure 1</a>.</p

Protein-protein interactions of PSI.

<p>A) GST pulldown assay using PSI mutant proteins. GST-PSI fusion proteins carrying the serine to alanine PSI mutations were phosphorylated using purified human casein kinase II and incubated with Kc nuclear extract. The resulting glutathione resin eluates were analyzed by silver staining and mass spectrometry. B) Silver stain of PSI and interacting proteins following anti-polyoma and anti-PSI immunoprecipitations. The asterisk indicates antibody heavy chain. C) Immunoblot analysis of (B) using anti-PSI antibody. D) Mass spectrometry analysis of (B). Proteins identified as interacting with PSI and the number of peptides observed for each protein are listed.</p

Purification of a second PSI kinase activity.

<p>A) Purification strategy for tousled-like (tlk) kinase. B) Protein composition of peak fraction activity from the final Mono S column visualized by silver staining. The species identified as tlk is labeled. C) <i>In vitro</i> kinase assay of PSI mutants. Serine to alanine PSI mutants were phosphorylated <i>in vitro</i> using the peak fraction of activity from the final Mono S column. Assays were visualized using autoradiography, and, to ensure equal protein loading, silver staining.</p