CELL CYCLE-DEPENDENT PHOSPHORYLATION OF MAMMALIAN PROTEIN PHOSPHATASE1 BY CDC2 KINASE

Protein phosphatase 1 (PP-1) is known to be a critical component of eu karyotic 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 phosp horylation of PP-1 in intact mammalian cells, an antibody has been pre pared 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 m id-mitosis. The phosphorylation of T320 of PP-1 mas reduced by the cyc lin-dependent protein kinase inhibitor, olomoucine, and increased by t he PP-1/PP-2A inhibitor, calyculin A, Immunofluorescence microscopy us ing phospho-T320 antibody indicated that in NIH 3T3 cells the phosphor ylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase, Immunostaining indicated that phospho-PP-1 was loc alized exclusively to nonchromosomal regions, Furthermore, in cell fra ctionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction, These observations suggest that phosphorylati on by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activ ity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division .