Cloning and characterization of a novel mammalian PP2C isozyme

PP2C is a structurally diversified protein phosphatase family with a wide r ange of functions in cellular signal transduction. A novel PP2C subtype, de signated PP2C delta, was identified from a rat cDNA clone, which encodes a protein of 392 amino acid residues. While PP2C delta shares approximately 3 0% sequence identity in its catalytic domain with the mammalian PP2C, it la cks a SO-residue carboxyl-terminal sequence conserved in mammalian PP2C. No rthern blot analysis showed that PP2C delta is widely expressed in rat tiss ues. The transcription of the PP2C delta gene was activated in response to stress, such as the addition of ethanol to the culture medium or UV irradia tion of cells. Recombinant PP2C delta purified from bacteria exhibited a po tent Mn2+-dependent serine/threonine phosphatase activity. Unlike other mem bers of the PP2C family, the activity of PP2C delta was inhibited, rather t han stimulated, by Mg2+. Transfection with PP2C delta resulted in inhibitio n of cell growth, precluding generation of stable 293 or CHO transfectants. Using a modified tetracycline-regulated PP2C delta-GFP dicistronic express ion cassette, it was revealed that overexpression of PP2C delta blocked cel l cycle progression and arrested cells at early S phase, resulting in inhib ition of DNA synthesis and leading to cell death. These results suggest tha t PP2C delta plays a role in regulation of cell cycle progression via depho sphorylation of its substrates whose appropriate phosphorylation states mig ht be crucial for cell proliferation.