Protein phosphatase-1 activation and association with the retinoblastoma protein in colcemid-induced apoptosis

Protein phosphatase-1 (PP1) is cell cycle regulated and potentially related to apoptosis. We studied PP1 in HeLa cells exposed to colcemid, which leads first to mitotic block, then to cell death within 72 h. The soluble PP1 activity, which was low at 14 h (mitosis), was then reversibly activated (maximally around 48 h), with parallel changes in the protein levels of the alpha, gamma1 and delta PP1 isoforms. PP1 activation suggested its involvement in dephosphorylating proteins relevant to apoptosis. Among these, we examined the retinoblastoma protein (pRb). This was found hyperphosphorylated at 14 h. Hypophosphorylated pRb appeared at 24 h, increased at 48 h, and was the only form left at 72 h. PP1 was found to associate with immunoprecipitated pRb, as indicated by PP1 activity assays on the pRb-immunocomplexes. The pRb-associated PP1 activity was low at 14 h, maximal at 24 h, low again by 72 h and was due to PP1delta. The presence of active PP1 suggests its involvement in pRb dephosphorylation.