Phosphorylation-induced Conformational Changes in the Retinoblastoma Protein Inhibit E2F Transactivation Domain Binding*

Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2FTD) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2FTD binding ∼250-fold and that phosphorylation at Ser608/Ser612 and Thr356/Thr373 is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2FTD binding. We find that phosphorylation at Ser608/Ser612 promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2FTD binding site. We also find that phosphorylation of Thr356/Thr373 inhibits E2FTD binding in a manner that requires the Rb N-terminal domain. Taken together, our results suggest two distinct mechanisms for how phosphorylation of Rb modulates association between E2FTD and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.

The retinoblastoma tumor suppressor protein (Rb) 2 is a key negative regulator of cell proliferation, and Rb pathway deregulation is ubiquitous in cancer (1,2). Rb is inactivated by cyclindependent kinases (Cdk) in response to positive growth signals, which results in cell cycle progression (3)(4)(5)(6). Rb function as a growth inhibitor in part depends on its ability to repress the transcription activity of E2F (7)(8)(9)(10). E2F expression or Rb inactivation induces S phase entry, whereas Rb expression arrests cells in G 1 ; these observations directly implicate the Rb-E2F pathway as an essential control mechanism of the G 1 -S transition and a critical link between growth factor signaling and cell cycle progression (1,2). In quiescent cells and early G 1 , Rb is hypophosphorylated and bound to E2F in a manner that inhibits transactivation. Phosphorylation of Rb by both Cdk4/6-cyclin D and Cdk2-cyclin E occurs in late G 1 and results in the dissociation of Rb-E2F complexes and E2F activation (4,5,(11)(12)(13)(14)(15). The importance of phosphorylation in Rb inactivation and cellular proliferation is emphasized by the fact that tumor cells often have alterations to upstream regulators that result in Rb hyperphosphorylation (1,2). However, the molecular basis for how phosphorylation inhibits E2F binding has not been established.
The Rb protein consists of a structured N-terminal domain (RbN) that associates with a structured central domain called the "pocket" (Fig. 1A) (16). The C-terminal domain (RbC) is intrinsically disordered (17). Two additional unstructured sequences exist; one is between RbN and the pocket, which we term the interdomain linker (RbIDL), and the other is a linker within the pocket domain (RbPL) that connects the two structured pocket subdomains (18). The Rb-E2F complex is stabilized by two distinct interactions, both of which have been shown to be necessary for growth suppression and inhibition of E2F transcription activity (19,20). The Rb pocket domain binds the E2F transactivation domain (E2F TD ) (21,22), whereas RbC associates with the so-called marked box domains of E2F and its heterodimerization partner DP (Fig. 1B) (17).
Human Rb contains 16 Cdk consensus phosphorylation sites, although only a subset of these sites have been found phosphorylated in cells (14). The serine/threonine phosphoacceptor sites are distributed throughout the protein and, with a few exceptions, are in regions of the protein that lack intrinsic structure (Fig. 1A). Cell-based assays to uncover the particular phosphorylation events that result in Rb-E2F dissociation and reversal of Rb growth suppression have shown that phosphorylation at many different sites is capable of inactivating Rb (11-13, 23, 24). Insights into the distinct molecular effects of these phosphorylations is therefore critical to understanding the significance of multiple, seemingly redundant pathways toward Rb-E2F dissociation. One possibility is that different phosphorylation events control the two separate Rb-E2F interactions. Indeed, we previously found that phosphorylation of sites in RbC induces an intramolecular interaction between RbC and the pocket domain that specifically blocks the RbC-marked box association (17). It has also been shown that phosphorylation of sites in the pocket domain is capable of reducing E2F TD binding (22).
Here, we identify unambiguously the key phosphorylation events and characterize the domain rearrangements in Rb that result in inhibition of the E2F TD -pocket domain association. Phosphorylation of Thr 356 /Thr 373 in RbIDL and Ser 608 /Ser 612 in RbPL are each sufficient for partial inhibition of E2F TD binding, but both are necessary for complete inhibition. We show that phosphorylation stimulates an intramolecular interaction between RbPL and the pocket domain that overlaps with the E2F TD binding site. Our data confirm a role for RbPL, RbIDL, and the structured RbN in Rb inactivation and provide the first molecular insights into how phosphorylation disrupts a key cell cycle and growth regulatory complex.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Human Rb constructs containing a single domain (e.g. RbN or pocket domain) could be expressed with high yields in Escherichia coli. Constructs containing multiple domains required expression in Sf9 cells to obtain quantities suitable for biophysical assays. Thus, Rb 55-928 , Rb 55-787 (wild type and mutants), and Rb 380 -928 were all expressed in Sf9 cells as His 6 fusion proteins. Cells were infected at a density of ϳ2 ϫ 10 6 /ml with baculovirus containing the desired gene and incubated for 2-3 days. Proteins were purified by Ni 2ϩ -nitrilotriacetic acid affinity purification and heparin sulfate chromatography. Rb 352-787 , Rb 380 -787 (wild type and mutants), and RbP ⌬PL (Rb 380 -787 , with 578 -642 deleted) were expressed in E. coli as glutathione S-transferase fusion proteins. Cells were induced overnight at room temperature. The proteins were purified with glutathione affinity chromatography, the glutathione S-transferase tag was cut off, and the Rb domain was isolated by heparin sulfate chromatography. RbPL 592-624 , Rb 338 -379 , Rb 55-379 , and E2F TD (E2F1, residues 372-437) were expressed as His 6 fusion proteins in E. coli. Cells were induced for 2-4 h at 37°C, and proteins were purified by Ni 2ϩ -nitrilotriacetic acid affinity and anion exchange chromatography. Isotopically labeled RbP ⌬PL and RbPL 592-624 were prepared for NMR as described, except that upon induction, E. coli were switched to M9 minimal medium including [ 15 N]ammonium chloride, [ 13 C]glucose, and D 2 O as necessary. PP1 catalytic domain (␣ isoform) was expressed in E. coli and purified with anion exchange and heparin sulfate chromatography. Recombinant Cdk6-CycK (herpesvirus cyclin) and Cdk2-CycA were expressed and purified as described previously (25,26).
Enzymatic Modifications-Rb protein constructs were concentrated to ϳ1-5 mg/ml following purification and then phosphorylated in a reaction containing 10 mM MgCl 2 , 10 mM ATP, 250 mM NaCl, 25 mM Tris (pH 8.0), and 2% Cdk6-CycK or 10% Cdk2-CycA (percentage of mass of the total substrate in the reaction). Reactions were incubated at room temperature for 1 h. Use of either kinase resulted in similar phosphate incorporation in the reaction described in supplemental Fig. 1. Kinase-treated Rb 55-928 was digested with either trypsin or chymotrypsin and analyzed for phosphate incorporation using a Thermo Finnigan liquid chromatography/MS/MS (LTQ) linear ion trap. All MS/MS spectra were processed using Bioworks 3.3. Peptide identifications with better than 0.01 peptide probability were accepted and manually inspected.
Phosphatase reactions were carried out with 10% PP1 (percentage of mass of substrate) in the presence of 1 mM MnCl 2 , 250 mM NaCl, 25 mM Tris (pH 8.0) at room temperature for 1 h. We have found by mass spectrometry and radioisotope labeling assays that these conditions lead to nearly quantitative dephosphorylation (data not shown). Initially, proteins were purified following the enzymatic treatment and prior to isothermal titration calorimetry (ITC) or NMR with size exclusion chromatography; subsequently it was found this step was not necessary because results were unaffected by the purification step.
ITC-ITC experiments were conducted with a MicroCal VP-ITC calorimeter. Typically, ϳ0.5-1 mM E2F TD and 25-50 M Rb were used in each experiment. Proteins were dialyzed overnight prior to the assay in a buffer containing 100 mM NaCl, 1 mM dithiothreitol, and 25 mM Tris (pH 8.0). Data were analyzed with the Origin calorimetry software package assuming a onesite binding model. n values, reflecting the stoichiometry of the Rb-E2F TD complex, were between 0.8 and 1.2. Experiments were repeated for each Rb construct 2-4 times, and the reported error is the S.D. of each set of measurements.

RbN Is Required for Phosphorylation-induced Inhibition of E2F TD Binding-
To determine the precise sequences and phosphorylation sites within Rb required for inhibition of E2F TD binding, we applied an ITC assay to quantitatively measure affinities with purified proteins. We first expressed in Sf9 insect cells an Rb construct containing amino acids 55-928 (Rb 55-928 ). Rb 55-928 contains all three domains of Rb and all 15 conserved Cdk consensus sites. Rb 55-928 is phosphorylated by endogenous Sf9 kinases (33), so Rb 55-928 was dephosphorylated with the Rb phosphatase PP1. As seen in Fig. 2A, the affinity of E2F TD for the PP1-treated protein (dephosRb 55-928 ) is K d ϭ 0.04 Ϯ 0.02 M. This value is comparable with the affinity of E2F TD for unphosphorylated pocket domain purified from bacteria (Rb 380 -787 ; K d ϭ 0.045 Ϯ 0.007 M), which is the Rb domain necessary and sufficient for E2F TD binding (21,22).
We next phosphorylated Rb 55-928 with recombinant Cdkcyclin using reaction conditions that result in quantitative phosphorylation of accessible Cdk consensus sites (supplemental Fig. 1). Phosphorylation was detected at 13 of 15 Cdk consensus sites using phosphopeptide mapping with liquid chromatography/MS/MS (Table 1). Notably, we did not detect phosphorylation at Ser 230 or Ser 567 despite the large quantity of purified kinase used in the reaction. Both sites are buried in structured domains and have not been observed to be phosphorylated in vivo (14,21,22,34). Calorimetric assays show that E2F TD binds to phosRb 55-928 with K d ϭ 11 Ϯ 3 M ( Fig. 2A), which is ϳ250-fold weaker than its association with dephosRb 55-928 or unphosphorylated Rb 380 -787 . This result supports a large body of experiments demonstrating loss of E2F binding to Rb upon Cdk phosphorylation (4,5,(11)(12)(13)(14)(15).
To identify which Rb domains are required for inhibiting E2F TD association, we carried out a series of ITC experiments using Rb truncation mutants that were phosphorylated in our recombinant Cdk reaction. The results of these assays are summarized in Fig. 2B, and sample ITC data are shown in supplemental Fig. 2. E2F TD binds an RbC truncation mutant (phosRb 55-787 ) with K d ϭ 13 Ϯ 3 M, indicating that deletion of RbC has no effect on the phosphorylation-induced change in E2F TD affinity. Therefore, association of phosRbC with the pocket domain is not necessary for inhibition of E2F TD binding, although it remains possible that it has a redundant effect. Part of RbC binds the E2F-DP marked box domains, and previous data demonstrate that RbC phosphorylation specifically inhibits that association (17).
Next, three different N-terminal domain truncation mutants (phosRb 380 -928 , phosRb 352-787 , and phosRb 380 -787 ) were used in binding assays. E2F TD binds significantly tighter to each of these phosphorylated constructs than to phosRb constructs containing the N-terminal domain (Fig. 2B). These differences in affinity demonstrate that RbN is required for the full inhibition of E2F TD binding that occurs upon Rb phosphorylation.
Phosphorylation of Thr 356 /Thr 373 and Ser 608 /Ser 612 Weakens E2F TD Binding to Rb 55-787 -To define further which phosphorylation events are necessary for inhibition of E2F TD -pocket binding, we purified Rb 55-787 constructs containing alanine mutations such that only specific sites were phosphorylated by kinase. E2F TD binds to wild-type dephosRb 55-787 with K d ϭ 0.3 Ϯ 0.2 M and phosRb 55-787 with K d ϭ 13 Ϯ 3 M (Table 2). Our mass spectrometry data indicate that within residues 55-787, Rb is phosphorylated at Ser 780 and three pairs of other Cdk consensus sites: Ser 249 /Thr 252 in RbN, Thr 356 /Thr 373 in RbIDL, and Ser 608 /Ser 612 in RbPL (Table 1). We purified and phosphorylated three mutant Rb 55-787 constructs, each with Ser 780 and one of the three pairs of phosphoacceptor sites left intact ( Table 2). The affinity of E2F TD for the phosRb 55-787 protein with the Thr 356 /Thr 373 and Ser 608 /Ser 612 sites mutated (K d ϭ 0.33 Ϯ 0.02 M) is equivalent to its affinity for wild-type dephosRb 55-787 . This measurement indicates that Cdk phosphorylation of Ser 249 , Thr 252 , and Ser 780 has no effect on E2F TD binding. The affinities of E2F TD for phosRb 55-787 with only either Ser 608 /Ser 612 /Ser 780 or Thr 356 /Thr 373 /Ser 780 intact were similar (K d ϭ 2.7 Ϯ 0.6 M and K d ϭ 2.9 Ϯ 0.1 M, respectively), and both are weaker than wild-type dephosRb 55-787 but stronger than wild-type phosRb 55-787 . Together these data demonstrate that phosphorylation of Thr 356 /Thr 373 and Ser 608 /Ser 612 can both partially inhibit E2F TD binding, but neither pair of phosphoacceptor sites is sufficient alone to reproduce the full inhibition observed upon phosphorylation of wild-type protein. To confirm that phosphorylation of both pairs of sites is together sufficient for inhibition, we generated phosRb 55-787⌬249/252 (only Ser 249 /Thr 252 mutated) and found that the affinity of E2F TD for this construct (K d ϭ 25 Ϯ 1 M) is similar to wild-type phosRb 55-787 (K d ϭ 13 Ϯ 3 M).
It is noteworthy that the E2F TD affinity for phosRb 55-787⌬249/252 is ϳ25-fold weaker than its affinity for phosRb 352-787 . Both constructs contain the necessary four phosphorylation sites (Thr 356 , Thr 373 , Ser 608 , and Ser 612 ); however, full inhibition of E2F TD binding was only observed for phosRb 55-787⌬249/252 , which contains RbN in addition to the required phosphorylation sites. This result suggests further the requirement of RbN for phosphorylation-induced inhibition of E2F, despite the fact that there is no requirement for phosphorylation of the RbN sites. We conclude that the structured RbN must be critical for the mechanism of inhibition.
It is also significant that phosphorylation of Ser 608 /Ser 612 causes similar inhibition in the absence of RbN (ϳ15-fold; compare in Fig. 2 K d values for Rb 380 -787 and phosRb 380 -787 ) and in the presence of RbN (ϳ9 fold; compare in Table 2 K d values for dephosRb 55-787 and phosRb 55-787⌬249/252/356/373 ). Therefore, the effect of Ser 608 /Ser 612 phosphorylation does not require RbN. In sum, our data demonstrate two distinct and independent mechanisms for E2F TD inhibition, each relying on phosphorylation of a specific pair of sites (Thr 356 /Thr 373 or Ser 608 /Ser 612 ).
Phosphorylation Mediates Binding of RbPL to the Rb Pocket Domain-Having identified the required phosphorylation events for E2F TD inhibition, we next explored the conforma-tional changes in Rb that cause inhibition. The calorimetry data indicate that the partial inhibition induced by phosphorylation of Ser 608 /Ser 612 does not require RbN. Ser 608 and Ser 612 are located in RbPL, which comprises a disordered stretch of amino acids linking the two pocket subdomains (Fig. 1A). We hypothesized that phosphorylation induces an intramolecular association between the phosphorylated sequence in the linker and the pocket in a manner analogous to that previously observed for phosRbC (17).
To test this model, we attempted to detect a weak interaction between isolated, phosphorylated RbPL and the pocket domain in trans. ITC experiments titrating phosRbPL into the Rb pocket domain did not yield any significant heat signal (data not shown). Alternatively, we applied an NMR assay that is more sensitive in detecting the anticipated weak intermolecular association. We first generated a uniformly 15 N-labeled phosRbPL 592-624 construct that contains the phosphorylation sites Ser 608 and Ser 612 as well as the only sequence of conserved amino acids within RbPL (Fig. 3A). The 1 H-15 N HSQC spectrum of phosRbPL 592-624 shows little chemical shift dispersion in the 1 H dimension; this observation is consistent with a lack of struc-ture in the pocket linker (Fig. 3B). We next purified a pocket domain construct (RbP ⌬PL ), previously used in crystallization experiments (21,22), in which the entire RbPL is deleted. Superposition of the HSQC spectrum of phosRbPL 592-624 alone (Fig. 3B, black) and a spectrum of phosRbPL 592-624 in the presence of unlabeled RbP ⌬PL (Fig. 3B, red) shows that chemical shift changes and peak broadening occur in the presence of Rb pocket. This observation is consistent with binding on the fast to intermediate exchange time scale and the increased correlation time of forming a complex. When the HSQC experiment is repeated using unphosphorylated RbPL 592-624 , no spectral changes are observed in the presence of RbP ⌬PL , indicating that the association is phosphorylationdependent (Fig. 3C).
phosRbPL Associates with the Pocket Domain at the E2F TD Binding Site-We next tested whether phosRbPL 592-624 and E2F TD directly compete for binding to the Rb pocket. Excess E2F TD was added to the sample containing both 15 N-labeled phosRbPL 592-624 and unlabeled RbP ⌬PL pocket. The resulting spectrum (Fig. 3D, red) shows reduced peak broadening compared with the spectrum taken without E2F TD (Fig. 3B, red). This observation is consistent with excess E2F TD displacing phosRbPL 592-624 from the pocket domain such that the spectrum in the presence of E2F TD resembles that of free phosRbPL 592-624 (Fig. 3D, black). phosRbPL 592-624 and E2F TD thus do not bind simultaneously to the pocket domain. The NMR and calorimetry data together demonstrate that RbPL phosphorylation induces a phosRbPL-pocket association that inhibits E2F TD binding.
Upon assigning the HSQC peaks to specific amino acids in RbPL 592-624 , comparison of peak broadening reveals that the effect is most dramatic for Thr 601 -Val 610 (Fig. 3B). Residues Asp 604 -Tyr 606 are well conserved in orthologs (Fig. 3A) and have some sequence similarity to residues Asp 425 -Phe 427 of the C terminus of E2F1 TD . The DLF sequence in E2F1 TD makes critical binding contacts to Phe 482 and Arg 467 in the pocket domain of Rb (Fig. 4A) (21). We supposed that phosRbPL also binds at this site in the pocket. To test this idea, we compared peak broadening in HSQC spectra of 15 N-labeled phosRbPL in the presence of unlabeled RbP ⌬PL , RbP ⌬PL/F482A , and RbP ⌬PL/R467A (Fig. 4). The RbP ⌬PL mutants do not induce the significant peak broadening that is observed with the wild type Rb pocket, suggesting that the affinity of the mutant constructs for phosRbPL is weaker. These data demonstrate that Phe 482 and Arg 467 are important for mediating binding between phosRbPL and the pocket and accordingly that the phosRbPL and E2F TD binding sites in the pocket domain overlap.    To confirm an important role for residues Asp 604 -Leu 607 in the inhibition of E2F TD binding, we tested the affinity of E2F TD for Rb 380 -787 mutants in which these residues were mutated to alanine individually or in combination (Table 3). E2F TD binds phosphorylated wild-type Rb 380 -787 (K d ϭ 0.7 Ϯ 0.4 M) with approximately 15-fold less affinity than unphosphorylated protein (K d ϭ 0.045 Ϯ 0.007 M). E2F TD binds all of the unphosphorylated mutant proteins in Table 3 with similar affinity as wild type (data not shown). We found that mutation of Met 605 had little effect on the affinity of E2F TD for phosphorylated Rb pocket, whereas mutation of Asp 604 , Tyr 606 , and Leu 607 each had a modest effect. Mutation of Asp 604 -Tyr 606 in combination produced an Rb pocket construct in which phosphorylation does not inhibit E2F TD binding significantly. We conclude that Asp 604 , Tyr 606 , and Leu 607 all probably contribute to the mechanism of E2F TD inhibition due to Rb phosphorylation at Ser 608 /Ser 612 .
Finally, we asked whether phosphorylation at Ser 608 and Ser 612 are both required for inhibiting binding between E2F TD and the Rb pocket. We constructed serine to alanine mutants for Ser 608 and Ser 612 separately and used ITC to quantify changes in E2F TD -phosRb 380 -787 binding ( Table 3). The phosphorylated S612A mutant has a similar affinity for E2F TD (K d ϭ 0.7 Ϯ 0.1 M) as wild type phosRb 380 -787 (K d ϭ 0.7 Ϯ 0.4 M), whereas the phosphorylated S608A mutant does not bind E2F TD as weakly (K d ϭ 0.15 Ϯ 0.01 M). These data indicate that Ser 608 phosphorylation is sufficient for E2F TD inhibition, whereas Ser 612 phosphorylation has only a modest effect.
The phosRbPL and phosRbN-RbIDL Binding Sites in the Pocket Domain Each Partially Overlap with the E2F TD Binding Site-Our ITC data demonstrate that RbN and phosphorylation at the RbIDL sites are together capable of inhibiting E2F TD binding. We examined whether phosRbN-RbIDL (phosRb 55-379 ) associates with the pocket by conducting NMR experiments that monitor signals from the pocket domain. A uniformly labeled 2 D-15 N sample of RbP ⌬PL results in a well resolved 1 H-15 N TROSY spectrum (supplemental Fig. 3) (35). We compared the effects of the addition of unlabeled (blue), respectively. D, resonance peak intensity ratios of phosRbPL in the presence of wild type RbP ⌬PL (black) and mutants R467A (red) and F482A (blue). The ratio I/I 0 is defined as the peak intensity of phosRbPL in the presence of RbP ⌬PL (I) divided by the peak intensity of phosRbPL alone (I 0 ). These data demonstrate that Arg 467 and Phe 482 in the pocket domain are critical for binding phosRbPL as well as E2F TD .  MAY 21, 2010 • VOLUME 285 • NUMBER 21 phosRbN-RbIDL (Fig. 5A), phosRbPL (Fig. 5B), or E2F TD (Fig.  5C) on the spectrum (full spectra in supplemental Fig. 3). Titration of unlabeled phosRb 55-379 or phosRbPL results in peak broadening indicative of binding with an intermediate exchange time scale. The subset of peaks that broaden in each experiment is different, suggesting that the manner in which phosRbPL and the pocket domain associate is distinct from how phosRb 55-379 associates with the pocket. Interestingly, the peaks that broaden upon the addition of both phosRbN-RbIDL and phosRbPL also undergo chemical shift changes upon the addition of E2F TD (Fig. 5C). This observation suggests that the phosRbN-RbIDL and phosRbPL binding sites in the pocket domain partially overlap with the E2F TD binding site. Binding of both has an independent and additive effect toward E2F TD inhibition, as observed in the calorimetry experiments.

DISCUSSION
We have applied a calorimetry assay with purified proteins to identify unequivocally which phosphorylation events in Rb are capable of inhibiting E2F TD binding, and our data reveal two distinct mechanisms for this inhibition. In the first mechanism, Ser 608 /Ser 612 phosphorylation induces an intramolecular association between RbPL and the pocket domain. This association occludes the E2F TD binding site in the pocket such that both phosRbPL and E2F TD cannot bind simultaneously. These results mark a novel role for RbPL, which has previously been poorly characterized. Interestingly, both the Rb paralogs p107 and p130 contain linkers in their pocket domains that have a phosphorylation site within a similar sequence context. This homology suggests that a similar phosphorylation-induced structural change may be conserved in the pocket protein family.
The second mechanism for E2F TD inhibition requires both RbN and phosphorylation at sites in RbIDL. Previously, it has been reported that RbN and the Rb pocket domain associate in a manner that is phosphorylation-independent (16). Our data here suggest that RbN and phosRbIDL together bind to the pocket domain in a manner that partially overlaps the E2F TD binding site. Further structural studies are required to examine in detail how phosphorylation changes the interactions between RbN-RbIDL and the pocket.
A critical advantage of our analysis with recombinant proteins is complete control of the sites and extent of phosphorylation. Previous investigations aimed at identifying the critical phosphorylation events that regulate Rb-E2F binding had mixed results (11-13, 23, 24). These studies generally relied on transient transfections of mutagenized proteins in cancer cell lines. Thus, specific conclusions may have been influenced by substoichiometric degrees of phosphorylation at the acceptor sites that varied depending on the mutant and kinase. In an in vitro reaction with large quantities of recombinant kinase, we have achieved nearly quantitative phosphate incorporation, allowing unambiguous interpretation of the molecular effects of phosphorylation. Nevertheless, our results agree with and further explain several key observations from these previous cellular assays. Importantly, in transfection experiments assaying E2F binding, repression of E2F transcription, and growth suppression, cumulative mutation of multiple phosphoacceptor sites was required to abolish the effect of Cdk phosphory-lation (11,13,23,24). With previous results, our data reveal that multiple phosphorylation events (Thr 356 /Thr 373 , Ser 608 /Ser 612 , Ser 788 /Ser 795 , and Thr 821 /Thr 826 ) are capable of inhibiting one of the two interfaces stabilizing the overall complex (Fig. 1), and thus many different combinations of Cdk phosphorylation must all be sufficient to inactivate Rb by disrupting the complex.
Previous studies, which analyzed the effects of Rb phosphorylation at specific sites in cancer cell models, support our molecular characterization of Rb-E2F inhibition in several ways. First, phosphorylation reverses Rb repression of E2F-dependent transcription even if all of the RbC sites have been mutated (13,24). This observation points to a role for phosphorylation outside of RbC in specifically regulating the E2F transactivation domain and is consistent with our calorimetry data. Second, mutation of Ser 608 /Ser 612 in addition to all of the RbC sites results in an Rb construct that cannot be regulated by phosphorylation, directly demonstrating the importance of Ser 608 /Ser 612 phosphorylation in Rb inactivation (13). Third, Cdk phosphorylation cannot regulate Rb when RbN is deleted and RbC phosphorylation sites are mutated (13). Our data confirm that RbN is required for full phosphorylation-induced E2F TD inhibition. Interestingly, our calorimetry and NMR data suggest that RbN is not required for the partial E2F TD inhibition induced specifically by Ser 608 /Ser 612 phosphorylation. One possible explanation for this discrepancy is that the quantitative calorimetry assay used here is more sensitive than the cellular assays in detecting partial inhibition of E2F TD binding.
The implications of multiple possible phosphorylation pathways to E2F dissociation are intriguing. Different phosphorylation sites are probably preferentially phosphorylated by different Cdk-cyclins (34,36), and thus diverse upstream regulators can affect Rb-E2F stability. On the other hand, although the sites have seemingly redundant roles in combining to inhibit E2F binding, each phosphorylation event is unique in the structural change it induces in Rb. The resulting conformations can differentially influence interactions of Rb with other proteins. For example, the intramolecular association of phosRbC with the pocket domain upon Thr 821 /Thr 826 phosphorylation competes with the binding of LXCXE-containing proteins in addition to inhibiting the RbC-E2F marked box interface (12,17,37). Inhibition of the E2F TD -pocket association through Ser 608 / Ser 612 phosphorylation, however, would still permit LXCXE protein interactions. Thus, in generating distinct phosphorylated Rb structures, the different E2F inhibition mechanisms resulting from various phosphorylation pathways allow for multiple signaling outputs.