Protein phosphatase-1 alpha, gamma 1, and delta: changes in phosphorylation and activity in mitotic HeLa cells and in cells released from the mitotic block.
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 alpha, gamma 1, and delta were analyzed in mitotic (nocodazole-blocked) HeLa cells. Phosphorylation on threonine increased in gamma 1 and delta at mitosis. alpha 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 gamma 1 and delta, but not alpha, 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 alpha and delta only. On the other hand, the activity of gamma 1 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.
Puntoni
F
F
Department of Biomedicine, University of Pisa, Italy.
Villa-Moruzzi
E
E
D002453:Cell Cycle; D002467:Cell Nucleus / Q000201:enzymology; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D007158:Immunologic Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1
D007527:Isoenzymes; D010750:Phosphoproteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1