ASSOCIATION OF PROTEIN PHOSPHATASE-1-DELTA WITH THE RETINOBLASTOMA PROTEIN AND REVERSIBLE PHOSPHATASE ACTIVATION IN MITOTIC HELA-CELLS AND IN CELLS RELEASED FROM MITOSIS

The retinoblastoma gene product (pRb) is dephosphorylated at the exit from mitosis and protein phosphatase-1 (PP1) seems to be responsible f or such dephosphorylation. Three isoforms of PP1 exist in mammalian ce lls, alpha, gamma 1 and delta, with differential subcellular localizat ion and potentially different targeting subunits and functions. In ord er to identify which isoform dephosphorylates pRb, we used isoform-spe cific antibodies and analyzed the association of the PP1 isoforms with pRb in nocodazole-blocked (mitotic) HeLa cells and in cells released from the mitotic block (early G1). PP1 delta was found associated with the pRb immunoprecipitated from a mitotic cell extract, whereas neith er PP1 gamma 1 nor PP1 alpha were detected. In G1 cells progressively less pRb and of lower Mr was detected in anti-PP1 delta immunocomplexe s, and pRb had almost disappeared by 8 h. The PP1 associated with pRb was inactive at mitosis, but underwent a quick activation as cells exi ted from mitosis, with a peak at 1 h. Then the activity decreased prog ressively and disappeared by 8 h. [32P]labeled pRb, obtained from G2 c ells, was dephosphorylated ''in vitro'' by PP1 delta obtained from ear ly G1 cells. Altogether, the results indicated that PP1 delta associat ed with pRb and may be responsible for the phosphatase activity detect ed in the pRb complexes, supporting the hypothesis that PP1 delta may be the isoform that dephosphorylates pRb. (C) 1997 Academic Press.