Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr(72) is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr(72) antibody to establish Thr(72) phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser(120), Ser(121), and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr(72) kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr(72) kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr(72) phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr(72) I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.