Protein Phosphatase 1 Catalytic Subunit Isoforms from Alfalfa: Biochemical Characterization and cDNA Cloning

The catalytic subunit of protein phosphatase 1 (PP1c) was purified from an alfalfa (Medicago sativa) microcallus cell culture. The preparation was inhibited by rabbit muscle inhibitor-2 and okadaic acid and had a molecular mass of 35 kDa. Five distinct cDNAs termed MsPP1α, -β, -γ, -δ, and -ϵ were cloned from aM. sativasomatic embryo library. MsPP1α was identical to a cDNA reported earlier [A. Páy, M. Pirck, L. Bögre, H. Hirt, and E. Heberle-BorsMol. Gen. Genet.244, 176–182, 1994], while the others represented novel isoforms encoded by separate genes. The predicted amino acid sequences of MsPP1α, -β, -γ, -δ, and -ϵ were highly similar to each other and to other known PP1c sequences. The GST–MsPP1ß fusion protein expressed inEscherichia coliwas catalytically active and was inhibited by inhibitor-2 and okadaic acid. Affinity-purified polyclonal MsPP1antipeptide antibody detected a protein of 36 kDa in crude cell extracts. These results proved that the cDNA clone encoded an active PP1c which was very similar to the purified enzyme. The mRNA and protein concentrations of PP1c as well as the specific activity of protein phosphatase 1 did not change during the cell cycle in a synchronized alfalfa cell culture. On the other hand, the isoforms exhibited different steady-state mRNA levels in different plant organs suggesting tissue-specific functions.