Abstract
BackgroundThe product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1.ResultsThe requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal.ConclusionThe work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.
Highlights
The product of the retinoblastoma-susceptibility gene is a substrate for Protein Phosphatase 1 (PP1)
We investigated the association between product of the retinoblastoma-susceptibility gene (pRb) and the PP1 isoforms, as well as the presence of PP1 regulatory subunits associated with the pRbdirected PP1
We report that the entire pRb molecule is required for optimal in vitro interaction with the PP1 isoforms derived from cell extract and that this interaction is a direct one
Summary
The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). PP1 plays a key role in the regulation of cell cycle progression and is involved in other processes, including gene expression, muscle contraction, glycogen metabolism and neurotransmission [2,3]. Hypo-phosphorylated pRb complexes with and sequesters the E2F family of transcription factors, thereby preventing the transcription of genes required for S-phase entry [13]. In middle to late G1, phosphorylation of pRb by Cdks results in the release and activation of E2F and other pRb-bound transcription factors, which than activate the transcription of S-phase genes [14]. During the Mto-G1 transition, pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state [15,16,17,18]
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