Fostriecin, an antitumor antibiotic produced by Streptomyces pulveraceus, is a strong inhibitor of type 2A (PP2A; IC 50 3.2 nM) and a weak inhibitor of type 1 (PPl ; IC 50 131 uM) serine/threonine protein phosphatases. Fostriecin has no apparent effect on the activity of PP2B, and dose-inhibition studies conducted with whole cell homogenates indicate that fostriecin also inhibits the native forms of PPl and PP2A. Studies with recombinant PP1/PP2A chimeras indicate that okadaic acid and fostriecin have different binding sites.

Activating phosphorylation of cyclin-dependent protein kinases (CDKsEukaryotic cell cycle progression is controlled by the sequential activation and inactivation of cyclin-dependent protein kinases (CDKs). To coordinate the cell cycle machinery, extracellular and intracellular signals regulate CDK activities through a variety of mechanisms, including association with regulatory subunits (cyclins, inhibitors, and assembly factors), subcellular localization, transcriptional regulation, selective proteolysis, and reversible protein phosphorylation

We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C␤2, a novel PP2C␤ isoform, and to PP2C␣. PP2C␣ and PP2C␤2 co-purified with Mg 2؉ -dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C␣ and PP2C␤2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C␣ and PP2C␤2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.Eukaryotic cell cycle progression is driven by the ordered activation and inactivation of cyclin-dependent protein kinases (CDKs).

No abstract

In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1␣, PP1␦, PP1␥1, and PP1␥2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1␥1 that specifically inhibit PP1␥1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC 50 ϳ 50 nM) were then employed to elucidate the cellular functions of PP1␥1 during cell cycle progression. In A549 cells, the inhibition of PP1␥1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after ϳ36 -48 h, when PP1␥1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1␥1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.