title: Phosphodiesterases in the cell cycle
Creators: name: Sheppard, Catherine Louise
Publisher: name: University of Glasgow
Year: 2002
Subjects: subject: Biochemistry, cellular biology.

description: Phosphodiesterases (PDEs) are of central importance in the regulation of cyclic AMP (cAMP) signalling, since they mediate its degradation. Cyclic AMP and the downstream kinase, protein kinase A (PKA), are central to many cellular processes including cell proliferation, where they exert effects on the cell cycle machinery at a number of levels. The study of PDEs through the cell cycle in mammalian somatic cells has been sparse. Here, an investigation into the activities of different PDE isoforms through the cell cycle was undertaken. Reproducible methods for arresting Rat-1 fibroblasts in different stages of the cell cycle were developed. These were used to determine the profiles of PDE activity in the different phases of the cycle. It was found that, in Rat-1 cells, there was an increase in total PDE activity during mitosis. This was found to be largely attributable to PDE4 activity since it was sensitive to rolipram inhibition. Selective immunoprecipitation allowed the changes to be attributed to specific PDE4 subfamilies. In particular PDE4D3, when immunoprecipitated from mitotic lysates, exhibited the highest activity. This increased activity of PDE4D3 coincided with a fraction of the protein migrating as a double bandshift when analysed by SDS-PAGE and immunoblotting. Both the increased activity and the bandshifts occurring during mitosis were specific to mitosis and both disappeared within 2 h following a release from mitotic block. Both the increased activity and the bandshifts seen during mitosis were sensitive to the general kinase inhibitor, staurosporine, but insensitive to a range of other more specific inhibitors for kinases such as PKA, PKC, PI 3-kinase, ERK and Cdk2/ Cyclin B. They were also sensitive to treatment with alkaline phosphatase, and could be maintained following release from mitosis using concentrations of okadaic acid characteristic of protein phosphatase 1 inhibition. HPLC and mass spectrometric analysis of tryptically digested PDE4D3 phosphopeptides, four residues were identified as being highly phosphorylated by kinases that were active in mitotic lysate. Three of these residues Ser61, Ser75 and Ser239 are previously unknown phosphorylation sites. The fourth phosphorylated residue, Ser579, has previously been shown to be phosphorylated by ERK. When the activity of PKA in cell lysates was assayed, it was noted that in mitosis there was a fall in PKA activity that coincided with the increases in PDE4D3 activity. Upon rolipram-mediated inhibition of PDE4 in these mitotic cells a concomitant increase in PKA activity was noted. Furthermore, this inhibition of PDE4 in mitotic cells caused an increase in the rate at which the cells left mitosis. These observations suggest that the phosphorylation and activation of PDE4D3 in mitosis, by an as yet unidentified kinase, may lead to the decrease in PKLA activity. The inhibition of PDE4 may cause an increase in the rate at which cells leave mitosis, suggesting a specific regulatory role for PDE4D3 in the control of the transition of cells through mitosis.