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 Thr72 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-Thr72 antibody to establish Thr72 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, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 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.
Leach
Craig
C
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Shenolikar
Shirish
S
Brautigan
David L
DL
D000595:Amino Acid Sequence; D002453:Cell Cycle; D018385:Centrosome / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D000097847:Protein Phosphatase Inhibitory Proteins
D004791:Enzyme Inhibitors; D015739:Nocodazole; D011506:Proteins; D011994:Recombinant Proteins; D012694:Serine; D013912:Threonine; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins