Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle.
Protein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases. Here we used a phosphosite specific antibody to investigate the phosphorylation of I-2. We fractioned extracts from HeLa cells arrested with nocodazole and assayed for PXTP kinases using recombinant I-2. One major and two minor peaks of kinase activity were identified and the major peak contained both active MAPK and cdk1::cyclinB1, confirmed by immunoblotting. Cells released from a double thymidine block synchronously progressed through mitosis and immunoblotting revealed transient phosphorylation of endogenous I-2 in cells only during mitosis, and corresponding phosphorylation of histone H3 (Ser10) and PP1 (Thr320). Activation of cdk1::cyclinB1 was coincident with I-2 phosphorylation, but neither MAPK nor GSK3 were phosphorylated at this time, so we concluded that in living cells only cdk1::cyclinB1 phosphorylated the PXTP site in I-2. Immunofluorescent staining of cells with the PXTP phosphosite antibody revealed highly specific staining of mitotic cells prior to anaphase, at which point the staining disappeared. Thus, phosphorylation of I-2 is catalyzed by cdk1::cyclinB1 and staining with a specific antibody should prove useful as a selective marker of cells in the early stages of mitosis.
Li
Mingguang
M
Center for Cell Signaling and Department of Microbiology University of Virginia School of Medicine Charlottesville, VA 22908, USA.
Stefansson
Bjarki
B
Wang
Weiping
W
Schaefer
Erik M
EM
Brautigan
David L
DL
D020816:Amino Acid Motifs; D002455:Cell Division; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D000097847:Protein Phosphatase Inhibitory Proteins
D018844:Cyclin-Dependent Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D006657:Histones; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D011392:Proline; D054645:Protein Phosphatase 1; D011506:Proteins; D013912:Threonine; D013936:Thymidine; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins