Protein Phosphatase-1α, γ1, and δ: Changes in Phosphorylation and Activity in Mitotic HeLa Cells and in Cells Released from the Mitotic Block

Abstract

Protein phosphatase-1 is phosphorylated “in vitro” by cdc2-cyclin B (E. Villa-Moruzzi,FEBS Lett.304, 211–215, 1992). In the present study the phosphatase-1 isoforms α, γ1, and δ were analyzed in mitotic (nocodazole-blocked) HeLa cells. Phosphorylation on threonine increased in γ1 and δ at mitosis. α was phosphorylated only in mitotic cells and mainly on serine. Exposure of permeabilized mitotic cells to a peptide that inhibits cdc2 decreased the phosphorylation of the isoforms. Cell fractionation indicated that phosphatase-1 was over 90% inactivated and phosphorylated in the soluble, but not in the chromosomal fraction of mitotic cells. Immunoprecipitation from the mitotic soluble fraction indicated that only γ1 and δ, but not α, were inactivated. Altogether the data pointed to a correlation between phosphatase-1 inactivation and phosphorylation in mitotic cells. cdc2-cyclin B might be the kinase (or one of the kinases) that phosphorylates phosphatase-1. In cells released from the mitotic block, the phosphatase-1 activity in the soluble, but not in the nuclear fraction, increased progressively, reaching control values by 16 h. Immunoprecipitation indicated that the increase in activity was due to α and δ only. On the other hand, the activity of γ1 remained low, and this was also the only isoform that remained phosphorylated, though less than in mitotic cells. Also in the case of the cells released from mitosis, a correlation may exist between phosphorylation and inactivation of phosphatase-1. However, the regulation of phosphatase-1 is complex and may involve also regulatory subunits that are still unknown. Altogether, the results indicated the differential regulation of the phosphatase-1 isoforms both at mitosis and in G1 cells.