ABSTRACT
Polo-like kinase (Plk) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Plk protein levels are low or undetectable in terminally differentiated cells and tissues and its expression is strongly correlated with cell growth. Plk protein and enzymatic activity are regulated by multiple mechanisms during cell cycle progression. During G1 Plk levels are low but increasing amounts of protein are detected during S phase and the highest amounts during G2M. Transcription of Plk message is specifically repressed during G1 but that cannot entirely account for the rapid disappearance of Plk protein at the end of mitosis. In this report we show that Plk protein can be degraded in vitro by partially purified proteasomes and that specific proteasome inhibitors can block Plk protein degradation both in vitro and in vivo. We also detected high molecular weight polyubiquitinated forms of Plk by immunoprecipitation and immunoblotting and confirmed that Plk, like other mitotic regulators, is targeted for destruction at the end of mitosis through the ubiquitin-proteasome mediated degradation pathway.
Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Protein Kinases/metabolism , Ubiquitins/metabolism , Animals , Cell Cycle , Cell Cycle Proteins , Cell-Free System , Cysteine Proteinase Inhibitors/pharmacology , In Vitro Techniques , Mice , Mitosis , Proteasome Endopeptidase Complex , Protein Kinases/chemistry , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Reticulocytes/metabolism , Tumor Cells, Cultured , Polo-Like Kinase 1ABSTRACT
Previously, we demonstrated that expression of polo-like kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34cdc2. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34cdc2-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.
Subject(s)
Mitosis/physiology , Protein Kinases/metabolism , Amino Acid Sequence , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle/physiology , Cell Cycle Proteins , Enzyme Activation , HeLa Cells , Humans , Immune Sera , Mice , Molecular Sequence Data , Phosphorylation , Protein Kinases/immunology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Polo-Like Kinase 1ABSTRACT
We have cloned both human and murine complementary DNAs that are homologous to the Drosophila serine/threonine polo kinase and the recently cloned murine polo related kinase (PLK). Both the human and murine clones are about 2.1 kilobases with open reading frames of 1.8 kilobases, encoding proteins of 603 amino acids with a predicted size of 66 kilodaltons and an apparent size of 67 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. During embryonic development of the mouse, the mRNA was expressed in all tissues examined, whereas in adult tissues, expression was limited to thymus and ovaries. All cell lines examined also expressed mRNAs of similar size. Microinjection of in vitro transcribed sense mRNA into serum-starved murine NIH3T3 cells induced tritiated thymidine incorporation, whereas microinjection of antisense RNA into growing NIH3T3 cells blocked tritiated thymidine incorporation. When PC12 rat cells were induced to differentiate with nerve growth factor, gene expression of PLK was greatly reduced. Together, these results suggest that PLK expression is restricted to, and is perhaps required by, proliferating cells.
