calyculin-a and olomoucine

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl(-) channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl(-) channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34(cdc2)/cyclin B. ClC-2 channels were phosphorylated by p34(cdc2)/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a (632)Ser-to-Ala (S632A) mutation in the C-terminus, indicating that (632)Ser is a target of phosphorylation by p34(cdc2)/cyclin B. Injection of activated p34(cdc2)/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34(cdc2)/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 microM), an effect that was inhibited by calyculin A (5 nM) but not by okadaic acid (5 nM). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34(cdc2)/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34(cdc2)/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors.

Topics: Animals; Chloride Channels; CLC-2 Chloride Channels; Cyclin-Dependent Kinases; Enzyme Inhibitors; Female; Kinetics; Kinetin; Marine Toxins; Mitosis; Okadaic Acid; Oocytes; Oxazoles; Phosphorylation; Purines; Recombinant Proteins; Xenopus

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.

Topics: 3T3 Cells; Animals; Antibodies; CDC2 Protein Kinase; Cell Cycle; Cell Line; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Kinetin; Mammals; Marine Toxins; Mice; Mitosis; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Purines; Recombinant Proteins; Threonine; Transfection

Histone H1 is highly phosphorylated in mitotic HeLa cells, but is quickly dephosphorylated in vivo at the end of mitosis and in vitro following cell lysis. We show here that okadaic acid and microcystin-LR block the in vitro dephosphorylation of H1 and that they do so directly by inhibiting the histone H1 phosphatase rather than by some indirect mechanism. The concentrations of microcystin and okadaic acid required for inhibition strongly suggest that the histone H1 phosphatase is either PP1 or an unknown protein phosphatase with okadaic acid-sensitivity similar to PP1. The histone H1 phosphatase is predominantly located in chromosomes with at most one copy for every 86 nucleosomes. This tends to support its identification as PP1, since localization in mitotic chromosomes is a characteristic of PP1 but not of the other known okadaic acid-sensitive protein phosphatases. We also show that treatment of metaphase-arrested HeLa cells with staurosporine and olomoucine, inhibitors of p34cdc2 and other protein kinases, rapidly induces reassembly of interphase nuclei and dephosphorylation of histone H1 without chromosome segregation. This result indicates that protein kinase activity must remain elevated to maintain a mitotic block. Using this as a model system for the M- to G1-phase transition, we present evidence from inhibitor studies suggesting that the in vivo histone H1 phosphatase may be either PP1 or another phosphatase with similar okadaic acid-sensitivity, but not PP2A.

Topics: Cell Nucleus; Chromatin; Enzyme Inhibitors; HeLa Cells; Histones; Humans; Kinetin; Marine Toxins; Metaphase; Microcystins; Mitosis; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Purines; Staurosporine