Association of Protein Phosphatase-1&dgr; with the Retinoblastoma Protein and Reversible Phosphatase Activation in Mitotic HeLa Cells and in Cells Released from Mitosis

Author： Puntoni F. Villa-Moruzzi E.

Publisher： Elsevier

ISSN： 0006-291X

Source： Biochemical and Biophysical Research Communications, Vol.235, Iss.3, 1997-06, pp. : 704-708

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

The retinoblastoma gene product (pRb) is dephosphorylated at the exit from mitosis and protein phosphatase-1 (PP1) seems to be responsible for such dephosphorylation. Three isoforms of PP1 exist in mammalian cells, , &ggr;1 and &dgr;, with differential subcellular localization and potentially different targeting subunits and functions. In order to identify which isoform dephosphorylates pRb, we used isoform-specific 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&dgr; was found associated with the pRb immunoprecipitated from a mitotic cell extract, whereas neither PP1&ggr;1 nor PP1 were detected. In G1 cells progressively less pRb and of lower Mr was detected in anti-PP1&dgr; immunocomplexes, and pRb had almost disappeared by 8 h. The PP1 associated with pRb was inactive at mitosis, but underwent a quick activation as cells exited from mitosis, with a peak at 1 h. Then the activity decreased progressively and disappeared by 8 h. [32P]labeled pRb, obtained from G2 cells, was dephosphorylated “in vitro” by PP1&dgr; obtained from early G1 cells. Altogether, the results indicated that PP1&dgr; associated with pRb and may be responsible for the phosphatase activity detected in the pRb complexes, supporting the hypothesis that PP1&dgr; may be the isoform that dephosphorylates pRb.