RBP1 is phosphorylated on serines 864 and 1007 in vivo and in vitro by cyclin A/CDK2. A, shown is phosphoamino acid analysis of GST-RBP1 phosphorylated in vitro by cyclin A/CDK2 in the presence of [γ-³²P]ATP (left panel) or immunoprecipitated FLAG-RBP1 metabolically labeled with [³²P]orthophosphate in HEK293 cells in vivo (right panel). The positions of phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) are marked by arrows. B, tandem mass spectrometry spectra of FLAG-RBP1 immunoprecipitated from HEK293 cell lysates is shown. (i) RBP1 859–867 (parent ion, 583.8 m/z 2+) shows phosphorylation at S864. (ii) RBP1 1004–1017 (parent ion, 785.37 m/z) shows phosphorylation at S1007. (iii) RBP1 1103–1122 (parent ion, 711.0 m/z 3+) shows phosphorylation at Ser-1109. AMU, atomic mass units. C, purified recombinant His₆-RBP1^784–930 (left panel) and His₆-RBP1^937–1073 (right panel) and their corresponding alanine mutants His₆-RBP1^{784–930 (S864A)} and His₆-RBP1^{937–1073 (S1007A)} were incubated in the absence (−) or presence (+) of cyclin A/CDK2 and [γ-³²P]ATP. After SDS-PAGE, the proteins were visualized by autoradiography (upper panels) and Coomassie Blue (lower panels). D, shown is a schematic representation of the domain organization of RBP1 and the position of the three phosphorylation sites (underlined) identified by mass spectrometry. Ser-864 and -1007, which conform to CDK consensus phosphorylation sites, are marked by black circles, whereas a gray circle marks Ser-1109. The asterisk denotes phosphorylation sites identified on endogenous RBP1 in phosphoproteomic studies (56, 57). The LVCHE motif (amino acids 957–961) responsible for binding to the pocket of pRb is indicated. The Tudor, ARID, and Chromo domains and the repressor regions 1 and 2 of RBP1 are indicated. Repressor region 2 binds to SAP30.

CDK-mediated phosphorylation of RBP1 and pRb induces their dissociation in vitro and in vivo during G₀/G₁-S phase cell cycle progression. A, cyclin A/CDK2-mediated phosphorylation of RBP1 and pRb disrupts their association in vitro. Purified recombinant MBP-pRb^279–928 immobilized on amylose resin was either unphosphorylated (pRb^279–928, lanes 4 and 5) or phosphorylated with cyclin A/CDK2 (pRb^279–928-(P), lanes 6 and 7). The samples were then incubated with either unphosphorylated GST-RBP1 (RBP1, lanes 4 and 6) or cyclin A/CDK2-phosphorylated GST-RBP1 (RBP1-(P), lanes 5 and 7), and binding was performed as described under “Experimental Procedures.” The level of GST-RBP1 binding was assessed by immunoblotting with anti-GST antibody (upper panel). As controls, unphosphorylated (lane 2) or phosphorylated GST-RBP1 (lane 3) was incubated with empty amylose resin. To demonstrate that changes in association between RBP1 and pRb were phosphorylation-dependent, ATP was omitted from the phosphorylation reactions (lanes 4–7, lower panel). Lane 1 represents 10% of the input of GST-RBP1 in the binding reaction. B, cyclin A/CDK2-mediated phosphorylation disrupts preformed RBP1·pRb complex in vitro. Pre-formed MBP-pRb^279–928·GST-RBP1 complex immobilized on amylose resin was incubated in the absence (lane 1) or presence of cyclin A/CDK2 (lane 2) under phosphorylation conditions and then washed. Bound GST-RBP1 was assessed by immunoblotting with anti-GST antibody. C, the level of RBP1-associated with pRb decreases as cells progress from G₀/G₁ to S phase of the cell cycle. Upper panel, lysates from either exponentially growing (lane 1), G₀/G₁ phase-arrested with ICI 182780 (lane 2, ICI), or S phase (27 h after the addition of estradiol to ICI 182780-arrested cells) (lane 3, ICI + E2) MCF-7 cells were subjected to Western blotting with an anti-pRb antibody to demonstrate relative levels of hypophosphorylated and hyperphosphorylated pRb. Middle panel, FLAG-RBP1 was immunoprecipitated (IP) from either exponentially growing (lane 2), G₀/G₁ phase arrested with ICI 182780 (lane 3, ICI), or S phase (27 h after the addition of estradiol to ICI 182780-arrested cells) (lane 4, ICI + E2) MCF-7 cells. Lane 1 represents control vector transfected cells. The immunoprecipitates were assessed for the levels of FLAG-RBP1 and co-immunoprecipitating pRb by immunoblotting with the appropriate antibodies. Lower panel, the histograms represent the relative levels of pRb bound to the immunoprecipitated FLAG-RBP1 from G₀/G₁ arrested (ICI) or cells in S phase (ICI + E2). Error bars represent ± S.E. of three independent experiments.

RBP1 is phosphorylated by CDKs in vitro and in vivo in a cell cycle-dependent manner. A, recombinant GST-RBP1 or GST-pRb^773–928 were incubated with the indicated cyclin/CDKs in the presence of [γ-³²P]ATP, separated by SDS-PAGE, and analyzed by autoradiography. B, FLAG-RBP1 was immunoprecipitated from [³²P]orthophosphate labeled cells, incubated in the presence (lane 2) or absence (lane 3) of the CDK1/2 inhibitor Roscovitine (50 μm), separated by SDS-PAGE, and subjected to autoradiography (³²P) or immunoblotting (IB) with anti-FLAG antibody. Immunoprecipitation of cells transfected with empty vector was performed as control (lane 1). C, upper panel, shown is the kinetics of G₁-S phase progression of MCF-7 cells transfected with empty vector (dashed black line) or expressing wild-type RBP1 (solid black line) after cells were arrested in G₀/G₁ phase by ICI 182780 treatment and stimulated with estradiol to synchronously re-enter cell cycle. Cells were stained with BrdU and harvested at the indicated times to determine the percentage of BrdU-positive cells. Error bars represent ± S.E of three independent experiments. Lower panel, FLAG-RBP1 expressed in [³²P]orthophosphate-labeled MCF-7 cells, subjected to the same treatment as in the upper panel, was immunoprecipitated from G₀/G₁ phase-arrested cells (time = 0 h; lane 2, ICI) or at 27 h after estradiol addition when cells were in S phase (time = 27 h, lane 3, ICI + E2). The immunoprecipitates were separated by SDS-PAGE and subjected to autoradiography (³²P) or immunoblotting with anti-FLAG antibody (IB) to determine loading. Immunoprecipitation of cells transfected with empty vector was performed as control (lane 1). This experiment is representative of two independent experiments.

CDK-mediated phosphorylation of RBP1 does not impact on its interaction with SAP30 in vitro or affect associated SAP30·mSin3A·HDAC activity in vivo. A, cyclin A/CDK2-mediated phosphorylation of GST-RBP1 does not affect binding to SAP30 in vitro. Upper panel, GST-RBP1^784–1257 immobilized on glutathione-agarose was either unphosphorylated (lane 2) or phosphorylated with cyclin A/CDK2 (lane 3) and incubated with purified recombinant His₆-SAP30 to assess binding, as described under “Experimental Procedures.” The level of His₆-SAP30 binding was assessed by immunoblotting with anti-His antibody. As a control, His₆-SAP30 was incubated with empty glutathione agarose (lane 1). Lower panel, in vitro kinase assay confirming phosphorylation of purified GST-RBP1^784–1257 with cyclin A/CDK2 + [γ-³²P]ATP (lane 2). B, preformed GST-RBP1^784–1257·His₆-SAP30 complex was incubated in the absence (lane 1) or presence of cyclin A/CDK2 (lane 2) under phosphorylation conditions and then washed to remove unbound His₆-SAP30. The level of His₆-SAP30 bound to GST-RBP1^784–1257 was assessed by immunoblotting with anti-His antibody. C, the levels of RBP1-associated SAP30 and mSin3A do not change during G₀/G₁-S phase cell cycle progression. FLAG-RBP1 was immunoprecipitated from either exponentially growing (lane 1), G₀/G₁-arrested with ICI 182780 (lane 3, ICI), or S phase (27 h after the addition of estradiol to ICI 182780-arrested cells) (lane 4, ICI + E2) MCF-7 cells. The immunoprecipitates were assessed for the levels of FLAG-RBP1 and co-immunoprecipitating SAP30 and mSin3A by immunoblotting with the appropriate antibodies. Lane 2 represents control immunoprecipitates from cells transfected with empty vector. Lower panel, the histograms represent the relative levels of SAP30 (left) and mSin3A (right) bound to the immunoprecipitated FLAG-RBP1 from G₀/G₁-arrested (ICI) cells or cells in S phase (ICI + E2). Error bars represent ±S.E of three independent experiments. D, RBP1-associated HDAC activity does not change during progression from G₀/G₁ (ICI) into S phase (ICI + E2). FLAG-RBP1 was immunoprecipitated from MCF-7 cells arrested in G₀/G₁ or in S phase, as described in C. Immunoprecipitates were then incubated with [³H]acetylhistones in the absence or presence of the HDAC inhibitor trichostatin A (TSA, 3 μm) to perform HDAC assays, as described under “Experimental Procedures.” The graph represents the means ± S.E. of three independent experiments. Immunoprecipitates were subjected to immunoblotting with anti-FLAG antibody to demonstrate RBP1 loading (lower panel, lanes 2–4).

CDK-mediated cumulative phosphorylation of pRb and concurrent phosphorylation of RBP1 promotes their dissociation to mediate release of the SAP30·mSin3·HDAC complex. During G₀/G₁ phase, when CDK activity is low, the RBP1/pRb interaction mediates HDAC-dependent pRb inhibition on E2F transcription factors. As cells progress into the cell cycle, G₁ and S phase CDKs phosphorylate both pRb and RBP1 to disrupt their interaction, removing SAP30·mSin3·HDACs from pRb and E2F and thereby allowing transcription of genes necessary for S phase cell cycle progression.