Figure 2

Plk3 interacts and phosphorylates the p125 subunit of Pol δ. (a) Sf9 cells either infected with p125 baculovirus or both His₆-Plk3 and p125 baculoviruses for 48 h. Cell lysates were prepared from the infected cells, as well as from uninfected control cells. Ni-NTA resin, which was capable of binding His₆-Plk3, was added to each lysate. After thorough washing, proteins bound to Ni-NTA resin, along with cell lysates, were blotted for p125. (b, c) Various fragments of p125 were expressed as GST fusion proteins. Approximately equal amounts of purified proteins, as well as GST alone and casein, were used for in vitro kinase assays in the presence of purified His₆-Plk3. Kinase buffer alone was also used as a negative control. Reaction mixtures were fractionated on denaturing SDS–polyacrylamide gels, followed by autoradiography. Each number denotes a peptide fragment encompassing the particular region of p125 protein. (d) The phosphorylated band (arrow GST-1-110) as shown in (c) was excised from the gel and eluted. The eluted protein was subjected to two-dimensional peptide analysis. The letters C and E correspond to chromatography and electrophoresis, respectively, and the letter o denotes the origin. (e) Each of five serine/threonine residues in the GST-1-110 fragment was replaced individually with alanine in five mutants through site-directed mutagenesis. Mutant proteins were expressed and purified and the purified proteins were analysed on an SDS–polyacrylamide gel, which was stained with Coomasie Brilliant blue (f). Roughly equal amounts of the purified mutant proteins as shown in e, along with casein, were used for in vitro kinase assays in the presence of His₆-Plk3