PHOSPHORYLATION OF PROTEIN PHOSPHATASE-1 ISOFORMS BY CDC2 CYCLIN-B IN-VITRO

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Vil la-Moruzzi, FEES Lett 304: 211-215, 1992). In the present study we sho w that all the three Phosphatase-1 isoforms, alpha, gamma 1, delta, ar e phosphorylated by cdc2-cyclin B. Phosphorylation is specific for thi s kinase and involves a C-terminal Thr. This site is most likely Thr 3 20 in alpha (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in gamma 1, delta and in the testis-specific gamma 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-l purified from skeletal muscle, which is a mixture of the alpha, gamma 1 and delta i soforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphope ptide map. This is in agreement with the different sequences of the is oforms in the C-terminal regions and may be useful to distinguish the isoforms in extracts from metabolically-labelled cells. Our results su ggest that all the Phosphatase-l isoforms may be potentially regulated at M-phase.