PROTEIN PHOSPHATASE-1-ALPHA, PHOSPHATASE-GAMMA-1, AND PHOSPHATASE-DELTA - CHANGES IN PHOSPHORYLATION AND ACTIVITY IN MITOTIC HELA-CELLS ANDIN CELLS RELEASED FROM THE MITOTIC BLOCK

Protein phosphatase-1 is phosphorylated ''in vitro'' by cdcs-cyclin B (E. Villa-Moruzzi, FEES Lett. 304, 211-215, 1992). In the present stud y the phosphatase-1 isoforms alpha, gamma 1, and delta were analyzed i n mitotic (nocodazole-blocked) HeLa cells. Phosphorylation on threonin e 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 cdcs decreased the phosphoryl ation of the isoforms. Cell fractionation indicated that phosphatase-1 was over 90% inactivated and phosphorylated in the soluble, but not i n the chromosomal fraction of mitotic cells. Immunoprecipitation from the mitotic soluble fraction indicated that only gamma 1 and delta, bu t not alpha, were inactivated. Altogether the data pointed to a correl ation between phosphatase-1 inactivation and phosphorylation in mitoti c cells. cdc2-cyclin B might be the kinase (or one of the kinases) tha t phosphorylates phosphatase-1. In cells released from the mitotic blo ck, the phosphatase-1 activity in the soluble, but not in the nuclear fraction, increased progressively, reaching control values by 16 h. Im munoprecipitation indicated that the increase in activity was due to a lpha and delta only. On the other hand, the activity of gamma 1 remain ed low, and this was also the only isoform that remained phosphorylate d, though less than in mitotic cells. Also in the case of the cells re leased from mitosis, a correlation may exist between phosphorylation a nd inactivation of phosphatase-1. However, the regulation of phosphata se-1 is complex and may involve also regulatory subunits that are stil l unknown. Altogether, the results indicated the differential regulati on of the phosphatase-1 isoforms both at mitosis and in G1 cells. (C) 1997 Academic Press.