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DP1 Phosphorylation in Multimeric Complexes: Weaker Interaction with Cyclin A through the E2F1 Cyclin A Binding Domain Leads to More Efficient Phosphorylation Than Stronger Interaction through the p107 Cyclin A Binding Domain
Biochemical and Biophysical Research Communications 258, 596 – 604 (1999) Article ID bbrc.1999.0656, available online at http://www.idealibrary.com on
DP1 Phosphorylation in Multimeric Complexes: Weaker Interaction with Cyclin A through the E2F1 Cyclin A Binding Domain Leads to More Efficient Phosphorylation Than Stronger Interaction through the p107 Cyclin A Binding Domain Peter Guida and Liang Zhu 1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received March 22, 1999
Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does. © 1999 Academic Press
Cyclin-dependent kinases (Cdks), a large family of heterodimers of a cyclin subunit and a kinase subunit, are the central players in eukaryotic cell cycle progression (1). The major biochemical activity of these dimers is that of a protein serine/threonine kinase, and the phosphorylation of many key cell cycle regulatory proteins by Cdks is crucial for proper cell cycle regulation. An important question is how the various Cdks specifically and efficiently recognize their cellular substrates. 1 Corresponding author: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room U-519, Bronx, NY 10461. Fax: 718-430-8975. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Stable physical interaction between cyclin-dependent kinases and their substrates has been found to be an important determinant for specific and efficient phosphorylation since many substrates physically interact with their kinases. The retinoblastoma protein pRB is one of the best established substrates of Cdks. pRB is recognized by the cyclin D-dependent kinases through the LxCxE motif in D type cyclins (2, 3), which was first identified in viral oncoproteins that stably interact with pRB family proteins (4). A similar sequence (VxCxE) has also been identified in cyclin E to be responsible for pRB phosphorylation by cyclin E-Cdk2 (5). Many other phosphorylation substrates of cyclin A- and/or cyclin E-dependent kinases, including pRB family members p107 and p130 (6-10), E2F transcription factor family members E2F1, E2F2, and E2F3 (11-15), single-stranded DNA binding protein HSSB-p34/RPA (16, 17), NPAT (18), and the newly identified cyclin D-Cdk4/6 substrate DMP1 (19), all stably interact with the kinases. NPAT and DMP1 were in fact identified through their physical interaction with the kinases. Stable interaction with Cdks is also used by the p21Cip1/p27Kip1 family of kinase inhibitors, which themselves can be phosphorylated by the bound kinases (20-23). Stable physical interaction is not only important for recognition of substrates that directly contact the kinase, but also plays important roles in the phosphorylation of substrates that do not come into direct contact with the kinase but are in multimeric complexes with it. In this case, other proteins serve as adaptors to provide docking sites for both the kinase and the substrate. This situation is perhaps best demonstrated by the cyclin A-Cdk2/p107/E2F4-DP1 complex and the cyclin A-Cdk2/E2F1-DP1 complex. The conserved interacting sequences in p107 (6) and E2F1 (12) mediate interaction with cyclin A-Cdk2,
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which can then phosphorylate other proteins in the complexes. For example, cyclin A-Cdk2 phosphorylates the DP1 that is indirectly connected to it in the cyclin A-Cdk2/E2F1-DP1 multimeric complex (12, 13), and this phosphorylation is important for proper cell cycle regulation (11). Although stable physical interaction is clearly important for target determination, the relationship between the stability of interaction and the efficiency of phosphorylation remains to be tested. It is widely assumed that a more efficient interaction leads to a greater extent of phosphorylation. However, it is difficult to determine this relationship by comparing various kinase-substrate pairs since different phosphorylation sites on different substrates can contribute to the efficiency of phosphorylation. In this report, we studied the relationship between stability of interaction and efficiency of phosphorylation by examining the phosphorylation of the same cell cycle regulatory protein (DP1) in different cyclin A-Cdk2 containing complexes. We chose DP1 because it is present in several cyclin A-Cdk2 containing complexes but does not interact directly with cyclin A as described above. We also wished to have the DP1 in different complexes interact with similar adaptor proteins, and wanted to be able to recover the different complexes with the same antibody. It was shown in vitro that cyclin A-mediated phosphorylation and regulation could be transferred to E2F4 via the fusion of the E2F1 cyclin A binding domain to the N-terminus of E2F4 (the E2F41A protein) (13). Therefore, we also studied the cyclin A-Cdk2/ E2F41A-DP1 complex because this complex and the cyclin A-Cdk2/p107/E2F4-DP1 complex provide an ideal pair for determining the relationship between the stability of cyclin A interaction and the efficiency of DP1 phosphorylation. In addition, the functional consequences of DP1 phosphorylation by cyclin A-Cdk2 were examined. Finally, since E2F4 is predominantly localized in the cytoplasm while the cyclin A binding domain of E2F1 also serves as a nuclear localization signal (24), the effects of forced nuclear localization of E2F4 were determined. We show that although the adaptor proteins are required to attach cyclin A-Cdk2 to the complexes with DP1, a more stable cyclin A interaction does not translate into a more efficient phosphorylation of DP1 in multimeric complexes. In fact, a weaker interaction led to a more efficient phosphorylation. This phosphorylation results in the functional down-regulation of the transactivation activity of the E2F41A-DP1 heterodimer, and this effect is not caused by the change in the cellular localization of E2F4. Mechanisms other than the stability of protein-protein interaction must exist to determine the efficiency of phosphorylation after the kinase is tethered into the complexes.
MATERIALS AND METHODS Plasmids. Plasmids Rc/CMV-E2F4, Rc/CMV-E2F41A, and Rc/ CMV-E2F41Adel24 were constructed by cloning the HindIII-to-XbaI fragments from the corresponding pcDNA1 plasmids (listed below) into the Rc/CMV vector (Invitrogen). pcDNA1-E2F4 was a gift from Doron Ginsberg and David Livingston (25). pcDNA1-E2F41A and pcDNA1-E2F41Adel24 were constructed by inserting PCR amplified BamHI-to-BglII fragments coding for the cyclin A binding domain of E2F1 or its del24 derivative (13) in-frame into the BamHI site of pcDNA1-E2F4. pcDNA3-HA-E2F-4.NLS (26), pCMV-HA-DP1 (27), pcDNA1-HADP1 and pcDNA1-HA-DP1(DP) (11), pCMV-cyclin A (28), pCMVCdk2 and pCMV-Cdk2DN (29), pCMV-p107 and pCMV-p107L19 (30), and pE2F-TK-Luc (31) have been described previously. Cell culture and transient transfection. Human osteosarcoma (U2OS) cells were grown in 5% CO 2 in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum (Gemini BioProducts) in 10 cm plates unless stated otherwise. Cells were transfected by calcium-phosphate methods (32). The following amounts of plasmid DNA were used in transfections (except for luciferase assays, which are described below): E2Fs: 11 ug; DP1: 11 ug; cyclin A: 10 ug; Cdk2: 5.3 ug; Cdk2DN: 5.3 ug; p107: 10 ug; and p107L19: 10 ug. Empty vector DNA was used at amounts equivalent to the other samples for control. For all assays, cells were harvested 42-48 hours after transfection. Immunoprecipitation and Western blotting. Cells were lysed in the presence of protease inhibitors (33) for 30 minutes on ice and cell debris was pelleted. Supernatants were immunoprecipitated for 60 minutes with the indicated antibodies that had been covalently coupled to protein A sepharose. After washing with ELB, samples were boiled in Laemmli buffer and resolved by SDS-PAGE. For Western blotting, proteins were transferred to Immobilon membrane (Millipore) and probed with the indicated antibodies. Anti-DP1 (WTH 16), anti-E2F4 (WUF 10 and 23), anti-E2F1 (KH 20 and 95) and antip107 (SD4) were provided by Ed Harlow. Anti-cyclin A (H-432) was purchased from Santa Cruz Biotechnology. HRP-conjugated mouse or rabbit IgG was used as secondary antibody (Amersham) and detection was performed by ECL (Amersham). Luciferase assays. Cells were seeded into 6-well plates and transfected with the following: 3 ug of E2F-TK-Luc reporter, 110 ng of E2F expression plasmid, 110 ng of DP1, and either 4.4 ug of vector plasmid or 3.3 ug of cyclin A plus 1.1 ug of Cdk2. For the samples lacking E2F4 and DP1, 220 ng of additional vector plasmid was substituted. Separate transfections were done to generate duplicates. Luciferase assays were performed as previously described (34), using D-luciferin substrate (Molecular Probes). Light output was measured for 10 seconds in a Berthold AutoLumat LB 953.
RESULTS We chose DP1 in multimeric complexes to study the relationship between the stability of cyclin A-Cdk2 interaction and the efficiency of phosphorylation. To assemble the various multimeric protein complexes pictured in Figure 1A in vivo, we overexpressed each of the components in U2OS cells through transient transfection. The degree of cyclin A association with each complex was determined by its co-immunoprecipitation with the E2F subunit as shown in Figure 1B. Immunoprecipitation of endogenous and overproduced wild type E2F4 brought down a similar, detectable amount of cyclin A. Since E2F4 does not contain cyclin A binding sequences found in E2F1 and therefore does
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FIG. 1. (A) E2F1 can bind directly to cyclin A, while E2F4 cannot. E2F4 can indirectly associate with cyclin A when it is tethered to it by the p107 protein. Fusion of E2F1’s cyclin A-binding domain onto the N-terminus of E2F4 enables the resulting fusion protein (E2F41A) to bind directly to cyclin A. Mutants E2F1del24 and E2F41Adel24 (lacking 24 amino acids within the cyclin A binding domain) were studied in parallel. (B) U2OS cells were transiently transfected with the indicated expression plasmids and whole cell lysates subjected to immunoprecipitation and Western blotting with the indicated antibodies to determine the levels of association with cyclin A.
not bind cyclin A directly (13), this association is largely through the endogenous p107 protein since immunodepletion of endogenous p107 before E2F4 immunoprecipitation greatly reduced cyclin A association with E2F4 (data not shown). In the same assay, E2F41A associated with about three to five fold more cyclin A than wild type E2F4, demonstrating the contribution of the E2F1 cyclin A binding domain. The
level of cyclin A association with E2F41A was similar to the level of association between E2F1 and cyclin A after the overexpression of E2F1, although in this case a different antibody was used for immunoprecipitation. The corresponding control mutants E2F1del24 and E2F41Adel24, which lacked 24 essential residues within the cyclin A binding domain, bound much less cyclin A.
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FIG. 2. After transient transfection of the indicated expression plasmids into U2OS cells, whole cell lysates were immunoprecipitated and Western blotted with the indicated antibodies to examine the E2F-DP1 associations.
Formation of the cyclin A-Cdk2/p107/E2F4-DP1 complex through co-expression of p107 enabled E2F4 to associate with a much greater amount of cyclin A than E2F41A (Figure 1B). The p107-dependent nature of this was verified by overexpressing L19, a p107 mutant able to bind cyclin A but unable to bind E2F (30), which restored the level of cyclin A association back to that of E2F4 with no added p107. Immunoprecipitation with an anti-p107 antibody brought down large amounts of cyclin A in both p107 and L19 transfected cells. The levels of the overexpressed E2F4, E2F1, and cyclin A proteins were comparable to themselves in all samples (data not shown). Thus, the association of cyclin A with the E2F1-DP1 complex or the E2F41A-DP1 complex was of much lower stability than its association with the p107/E2F4-DP1 complex. The dramatic difference seen in the latter two complexes was directly comparable since the two complexes were recovered through immunoprecipitation with the same antibody. Since phosphorylation of DP1 could be affected by its interaction with E2F1 or E2F4 in these complexes, we recovered DP1 through its co-immunoprecipitation with E2F. As shown in Figure 2, E2F4, E2F41A, and E2F41Adel24 in various E2F4-DP1 complexes and E2F4 in p107/E2F4-DP1 complexes all associated with DP1 to a similar extent as determined by immunoprecipitation and Western blotting. Immunoprecipitation with an anti-E2F1 antibody also brought down comparable levels of DP1 from the E2F1-DP1 complexes. While DP1 was present in all of these complexes, a clear difference in the electromobility patterns of the associated DP1 proteins was observed. DP1 associated with E2F1 and with E2F41A displayed an upward smear which was not noticeable when it was associated with E2F1del24, E2F4, E2F41Adel24, or E2F4 in the p107-containing complexes. To determine whether this type of migration pattern was due to hyper-phosphorylation, we treated the antiE2F4 immunoprecipitates with lambda protein phos-
phatase. The phosphatase treatment compressed the upward smear of E2F41A-associated DP1 (data not shown). We also performed this experiment in the presence of phosphatase inhibitors, which restored the banding pattern of the E2F41A-associated DP1 to that seen when only a mock phosphatase treatment was done (data not shown). Thus, the upward smear pattern of E2F41A-associated DP1 was due to hyperphosphorylation. To test whether the DP1 hyper-phosphorylation was dependent on Cdk2, we utilized a kinase-inactive mutant of this protein, referred to as Cdk2DN (29). When E2F41A and DP1 were co-expressed with Cdk2DN, the smeared banding pattern of the E2F41Aassociated DP1 protein was not observed, indicating that Cdk2 activity was responsible for the E2F41Amediated phosphorylation of DP1 (Figure 3A). The two forms of Cdk2 proteins complexed with the corresponding E2F4 proteins to a similar extent (data not shown). Consistent with the increased binding of cyclin A shown in Figure 1B, E2F41A associated with a greater amount of Cdk2 (both WT and DN) than E2F4 did (data not shown). Endogenous cyclin A-Cdk2 proteins produced an intermediate extent of E2F41Aassociated DP1 phosphorylation, between those resulting from overexpression of cyclin A-Cdk2 and of cyclin A-Cdk2DN (data not shown). Recently, the N-terminus of E2F1 has been shown to be responsible for the nuclear localization of E2F1, in addition to cyclin A binding (24). While E2F1-DP1 is nuclear, overexpressed E2F4-DP1 is localized in the cytoplasm (26, 35, 36). Consistent with these reports, immunofluorescence studies with an anti-E2F4 antibody indicated that E2F4 was cytoplasmic, but E2F41A was nuclear (data not shown). E2F1 is more potent in transactivation and promotion of S phase entry than E2F4, and it was shown that all of the functional differences between E2F1 and E2F4 were attributable to the different cellular localization of these two E2Fs. By the generation of chimeras, it was
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FIG. 3. U2OS cells were transiently transfected with the indicated expression plasmids, including a dominant negative version of Cdk2 (Cdk2DN) and a nuclear localized mutant of E2F4 (E2F4.NLS). Whole cell lysates were immunoprecipitated and analyzed by Western blotting with the indicated antibodies to (A) assess the role of Cdk2 activity in mediating DP1 hyper-phosphorylation, and to (B) determine the role of nuclear localization of E2F4 in mediating association with cyclin A and hyper-phosphorylation of DP1.
shown that the N-terminus of E2F1 (residues 1 to 126, containing the E2F1 cyclin A binding domain), or even the SV40 T antigen nuclear localization sequence, was sufficient to transport E2F4 into the nucleus and stimulate E2F4 activity in transactivation and S phase promotion assays (24). We therefore determined whether nuclear localization of E2F41A was responsible for its effects on DP1 phosphorylation. For this purpose, an E2F4 protein fused with the nuclear localization sequence of SV40 T antigen (E2F4.NLS, (26)) was compared with E2F41A. E2F4.NLS was co-transfected with DP1 and cyclin A-Cdk2 in parallel with E2F4 and E2F41A. Cell extracts were immunoprecipitated with an anti-E2F4 antibody, and the interaction with cyclin A and the phosphorylation status of co-precipitated DP1 were determined as before. Figure 3B shows that E2F4.NLS overexpression did not increase the interaction with cyclin A over that of E2F4, in spite of the similarity between the SV40 T antigen NLS (PKKKRKV) and the E2F1 cyclin A binding domain (PVKRRLDL) (12, 37). Overexpression of E2F4.NLS also did not promote DP1
hyper-phosphorylation like E2F41A, as indicated by the lack of the upward smear of the associated DP1 (Figure 3B). Protein expression levels of E2F4.NLS were comparable with that of E2F41A (data not shown). Therefore, simple nuclear localization without cyclin A interaction is not sufficient for efficient DP1 phosphorylation. This result is in fact consistent with our results obtained from the cyclin A-Cdk2/p107/ E2F4-DP1 complex. Although E2F4 is nuclear when associated with p107 ((26, 35) and data not shown), the formation of cyclin A-Cdk2/p107/E2F4-DP1 complexes did not result in hyper-phosphorylation of this E2F4associated DP1 (Figure 2). We investigated the transactivation potentials of E2F4-DP1 and E2F41A-DP1 in the presence and absence of exogenous cyclin A-Cdk2. After transient transfection of an E2F site-containing luciferase reporter plasmid and the indicated expression constructs, whole cell extracts were assayed for luciferase activity. E2F4-DP1, E2F41A-DP1, and E2F41Adel24DP1 all displayed similar transactivation activities (Figure 4A) under the conditions used in these exper-
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FIG. 4. (A) U2OS cells were transiently transfected with the indicated expression plasmids, along with an E2F-luciferase reporter plasmid. All transfections were performed in duplicate. Whole cell extracts were analyzed for luciferase activity over a 10 sec. time period. The values depicted in the graphs are the averages of duplicates, and the error bars show the deviation from the average value. The luciferase activity of E2F4-DP1 in each experiment was arbitrarily set to a value of 1.0, and was consistently 8-10 fold higher than the activity derived from reporter only-transfected extracts. The data shown are from representative experiments out of four. (B) After transient transfection of the indicated expression plasmids into U2OS cells, whole cell lysates were analyzed by immunoprecipitation and Western blotting with the indicated antibodies to assess the extent of phosphorylation of the E2F4-associated DP1 and DP1(DP) proteins. DP1(DP) contains a total of five serine/threonine to alanine mutations, and is recognized by the monoclonal antibody raised against DP1. Luciferase assays were performed as described in part (A). Shown are the data from a representative experiment out of three.
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iments. However, co-expression of cyclin A-Cdk2 revealed significant differences among their regulation. The transactivation activities of E2F4-DP1 and E2F41Adel24-DP1 were significantly stimulated by the inclusion of cyclin A-Cdk2 (about 3.5 fold each), likely due, at least in part, to the hyper-phosphorylation of cellular pocket proteins by the overexpressed cyclin A-Cdk2 and the consequent release of E2F from pocket protein-mediated repression (38). Indeed, expression of cyclin A-Cdk2 alone stimulated the E2F reporter activity (about 2 fold) over the vector control (data not shown). However, the stimulatory effect of cyclin A-Cdk2 on E2F4-DP1 was not observed on E2F41A-DP1, indicating that the tethering of cyclin A-Cdk2 onto E2F4 resulted in negative regulation. It was shown that the transactivation activity of E2F1 was down-regulated by cyclin A-Cdk2, dependent upon the stable interaction between E2F1 and cyclin A (12). Our results show that direct binding of cyclin A-Cdk2 to E2F41A can serve to down-regulate the transactivation activity of E2F4 as well. Nuclear localization of E2F4 through the SV40 T antigen nuclear localization sequence (the E2F4.NLS protein) again was not sufficient to produce the effects observed from E2F41A (Figure 4A). The activity of E2F4.NLS-DP1 was higher than that of E2F4-DP1 in the absence of transfected cyclin A-Cdk2. In addition, co-transfected cyclin A-Cdk2 strongly stimulated the transactivation activity of E2F4.NLS-DP1 (about 4 fold), further demonstrating the functional differences between E2F41A and E2F4.NLS. To test whether hyper-phosphorylation of DP1 in association with E2F41A was responsible for the observed functional down-regulation, we tested DP1(DP) in these assays. DP1(DP) is a mouse DP1 with Ala subtitutions in five potential Ser and Thr phosphorylation sites. It displayed diminished ability to be phosphorylated in in vitro kinase assays and produced S phase accumulation in cells infected with DP1(DP)-expressing viruses (11). Although this mutant still retained one potential phosphorylation site, it was clear from Figure 4B that it could not be hyper-phosphorylated by cyclin A-Cdk2 in association with E2F41A in vivo. In the transactivation assay, the activity of E2F41A-DP1(DP) was not significantly down-regulated by cyclin A-Cdk2, compared with DP1 in the same experiment (Figure 4B). Since the original DP1(DP) construct was based on vector pcDNA1, we also used pcDNA1-DP1 in these experiments, which consistently showed less activation by co-transfected cyclin A-Cdk2. These results confirm that the observed functional downregulation of E2F41A-DP1 was through the hyperphosphorylation of DP1 by the weakly associated cyclin A-Cdk2.
DISCUSSION In addition to the short consensus sequence flanking the phosphoacceptor site (S/T-P-polar residue-basic residue), stable protein-protein interaction has been found to be an important mechanism for substrate recognition by cyclin-dependent kinases (Cdks). The interaction can exist between the kinase and its substrate, or between the kinase and an adaptor molecule that in turn provides a docking site for the substrate. These two kinds of enzyme-substrate interactions are also important for other kinase-mediated regulations, such as JNK regulation of the jun family in response to extracellular signals (39). The presence in the cell of numerous multimeric complexes involving Cdks strongly indicates that indirect interaction through adaptor molecules is an important regulatory mechanism. For multimeric complexes, the nature and organization of adaptor proteins could be another important factor in determining the specificity and efficiency of regulation by the Cdks, in addition to stable interaction. The relative contributions of these two factors to the regulation have not been characterized, although it is usually assumed that stronger interactions are better for regulation since they can more efficiently increase the local concentration of the kinases. In this study, we have tested this assumption by comparing the phosphorylation of DP1 in the cyclin A-Cdk2/p107/ E2F4-DP1 complex and in the cyclin A-Cdk2/E2F41ADP1 complex. The results demonstrate that, while DP1 in the E2F4-DP1 dimer is not hyper-phosphorylated due to the lack of stable physical interaction between E2F4 and cyclin A, very stable cyclin A association through p107 also does not result in DP1 hyper-phosphorylation. On the other hand, weak association of cyclin A with E2F1, or with E2F41A, leads to efficient phosphorylation of DP1. Therefore, the stability of the interaction of cyclin A-Cdk2 with the complex does not dictate the phosphorylation efficiency of a substrate in the complex. The nature and structural organization of the adaptor proteins is important in determining the actual phosphorylation efficiency. It is conceivable that while certain subunit arrangement in the multimeric complex will help to bring the substrate closer to the kinase, other arrangement will actually restrict the access of the substrate to the kinase, even when the interaction of cyclin-Cdk with the complex is very stable. This might be the case with the cyclin A-Cdk2/ p107/E2F4-DP1 complex, in which p107 may only direct cyclin A-Cdk2 phosphorylation towards itself (7, 21, 40). Future structural studies of these multimeric complexes will provide physical bases for the targeting specificity in multimeric complexes. While the identification of consensus cyclininteracting sequences in many unrelated proteins pro-
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vides a structural explanation for enzyme-substrate interaction, at the same time it adds complexity to the issue of enzyme-substrate specificity. If most of the substrates use similar sequences to interact with cyclin-dependent kinases, how then is the specificity determined among these substrates? Use of different adaptor proteins can provide the specificity for substrates that indirectly interact with the kinase through the adaptors. This is perhaps best demonstrated by the F-box proteins that determine the target specificity of ubiquitin-mediated protein degradation in proteasomes. Different F-box proteins tether the same SCF ubiquitin-ligase complex to different target proteins (41). Our findings indicate the presence of a further level of specificity determination. After the Cdks are tethered to the complexes, the phosphorylation efficiency of a substrate in the complexes still needs to be determined by the nature of its adaptor proteins and the arrangement of the subunits in the complexes. Our results also reveal an important phenomenon regarding the regulation of E2F4-DP1 by the N-terminus of E2F1. A prominent difference among the E2F family members is their relationship with the cyclin A-dependent kinases. E2F1 contains a dedicated domain for direct association with cyclin A while E2F4 does not. Interestingly, this domain also serves as a nuclear localization sequence for E2F1, and the simple nuclear localization of E2F4 was shown to account for the functional differences between E2F1 and E2F4 (24). Our results show that the hyper-phosphorylation of E2F41A-associated DP1 and the down-regulation of the heterodimer’s transactivation activity are dependent on binding to cyclin A-Cdk2, not just on nuclear localization. Currently, studies are in progress to determine the effects on the cell cycle and on target gene expression by this kind of mis-regulation of E2F4. ACKNOWLEDGMENTS We thank Doron Ginsberg, Wilhelm Krek and David Livingston; Sander van den Heuvel and Ed Harlow; Philip Hinds and Robert Weinberg; and Richard Pestell for various reagents used in this study, and Anthony Karnezis for critical reading of the manuscript. We also thank the Pestell lab for assistance with luciferase assays. This work was supported by an American Cancer Society Research Project Grant 97-125-01. The Albert Einstein Cancer Center core support is also acknowledged. P.G. was supported by an NIH Cell and Molecular Biology Training Grant and L.Z. is a Leukemia Society of America Scholar.
REFERENCES 1. Morgan, D. D. (1997) Annu. Rev. Cell Dev. Biol. 13, 261–291. 2. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499 –511. 3. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J., and Livingston, D. M. (1993) Cell 73, 487– 497. 4. Ludlow, J. W., and Skuse, G. R. (1995) Virus Research 35, 113– 121.
5. Kelly, B. L., Wolfe, K. G., and Roberts, J. M. (1998) Proc. Natl. Acad. Aci. USA 95, 2535–2540. 6. Ewen, M., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85– 87. 7. Faha, B., Ewen, M., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87–90. 8. Lees, E., Faha, B. F., Dulic, V., Reed, S. I., and Harlow, E. (1992) Genes Dev. 6, 1874 –1885. 9. Li, Y., Graham, C., Lacy, S., Duncan, A. M. V., and Whyte, P. (1993) Genes Dev. 7, 2366 –2377. 10. Peeper, D. S., Parker, L. L., Ewen, M. E., Toebes, M., Hall, F. L., Xu, M., Zantema, A., van der Eb, A. J., and Piwnica-Worms, H. (1993) EMBO J. 12, 1947–54. 11. Krek, W., Xu, G., and Livingston, D. M. (1995) Cell 83, 1149 – 1158. 12. Krek, W., Ewen, M. E., Shirodkar, S. Z., Arany, Z., Kaelin, W. G., and Livingston, D.M. (1994) Cell 78, 161–172. 13. Dynlacht, B. D., Moberg, K., Lees, J. A., Harlow, E., and Zhu, L. (1997) Mol. Cell. Biol. 17, 3867–3875. 14. Dynlacht, B. D., Flores, O., Lees, J. A., and Harlow, E. (1994) Genes Dev. 8, 1772–1786. 15. Xu, M., Sheppard, K.-A., Peng, C.-Y., Yee, A. S., and PiwnicaWorms, H. (1994) Mol. Cell. Biol. 14, 8420 – 8431. 16. Dutta, A., and Stillman, B. (1992) EMBO J. 11, 2189 –2199. 17. Gibbs, E., Pan, Z. Q., Niu, H., and Hurwitz, J. (1996) J. Biol. Chem. 271, 22847–22854. 18. Zhao, J., Dynlacht, B., Imai, T., Hori, T., and Harlow, E. (1998) Genes Dev. 12, 456 – 461. 19. Hirai, H., and Sherr, C. J. (1996) Mol. Cell. Biol. 16, 6457– 6467. 20. Zhang, H., Hannon, G. J., and Beach, D. (1994) Genes Dev. 8, 1750 –1758. 21. Zhu, L., Harlow, E., and Dynlacht, B. D. (1995b) Genes Dev. 9, 1740 –1752. 22. Blain, S. W., Montalvo, E., and Massague, J. (1996) J. Biol. Chem. 272, 25863–25872. 23. Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M., and Clurman, B. E. (1997) Genes Dev. 11, 1464 –1478. 24. Muller, H., Moroni, M. C., Vigo, E., Petersen, B. O., Bartek, J., and Helin, K. (1997) Mol. Cell. Biol. 17, 5508 –5520. 25. Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z.-X., Xu, G., Wydner, K. L., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 2665–2679. 26. Lindeman, G. J., Gaubatz, S., Livingston, D. M., and Ginsberg, D. (1997) Proc. Natl. Acad. Sci. USA 94, 5095–5100. 27. Helin, K., Wu, C., Fattaey, A., Lees, J., Dynlacht, B., Ngwu, C., and Harlow, E. (1993) Genes Dev. 7, 1850 –1861. 28. Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Cell 70, 993–1006. 29. van den Heuvel, S., and Harlow, E. (1993) Science 262, 2050 – 2054. 30. Zhu, L., Enders, G., Lees, J. A., Beijersbergen, R. L., Bernards, R., and Harlow, E. (1995a) EMBO J. 14, 1904 –1913. 31. Pestell, R. G., Albanese, C., Lee, R. J., Watanabe, G., E., M., Johnson, J., and Jameson, J. L. (1996) Cell Growth Differ. 7, 1337–1344. 32. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1990) Current Protocols in Molecular Biology. Wiley Interscience, New York. 33. Jiang, H., Chou, H. S., and Zhu, L. (1998) Mol. Cell. Biol. 18, 5284 –5290. 34. Watanabe, G., Albanese, C., Lee, R. J., Reutens, A., Vairo, G.,
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Henglein, R., and Pestell, R. G. (1998) Mol. Cell. Biol. 18, 3212– 3222. 35. Magae, J., Wu, C. L., Illenye, S., Harlow, E., and Heintz, N. H. (1996) J. Cell Sci. 107, 1717–1726. 36. Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997) Mol. Cell. Biol. 17, 7268 –7282. 37. Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nallin, C. M., and Kaelin, J. W. G. (1996) Mol. Cell. Biol. 16, 6623– 6633.
38. Bremner, R., Cohen, B. L., Sopta, M., Hamel, P. A., Ingles, C. J., Gallie, B. L., and Phillips, R. A. (1995) Mol. Cell. Biol. 15, 3256 –3265. 39. Kallunki, T., Deng, T., Hibi, M., and Karin, M. (1996) Cell 87, 929 –939. 40. Woo, M. S., Sanchez, I., and Dynlacht, B. D. (1997) Mol. Cell. Biol. 17, 3566 –3579. 41. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J., and Harper, J. W. (1997) Cell 91, 209 –219.
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DP1 Phosphorylation in Multimeric Complexes: Weaker Interaction with Cyclin A through the E2F1 Cyclin A Binding Domain Leads to More Efficient Phosphorylation Than Stronger Interaction through the p107 Cyclin A Binding Domain Peter Guida and Liang Zhu 1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received March 22, 1999
Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does. © 1999 Academic Press
Cyclin-dependent kinases (Cdks), a large family of heterodimers of a cyclin subunit and a kinase subunit, are the central players in eukaryotic cell cycle progression (1). The major biochemical activity of these dimers is that of a protein serine/threonine kinase, and the phosphorylation of many key cell cycle regulatory proteins by Cdks is crucial for proper cell cycle regulation. An important question is how the various Cdks specifically and efficiently recognize their cellular substrates. 1 Corresponding author: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room U-519, Bronx, NY 10461. Fax: 718-430-8975. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Stable physical interaction between cyclin-dependent kinases and their substrates has been found to be an important determinant for specific and efficient phosphorylation since many substrates physically interact with their kinases. The retinoblastoma protein pRB is one of the best established substrates of Cdks. pRB is recognized by the cyclin D-dependent kinases through the LxCxE motif in D type cyclins (2, 3), which was first identified in viral oncoproteins that stably interact with pRB family proteins (4). A similar sequence (VxCxE) has also been identified in cyclin E to be responsible for pRB phosphorylation by cyclin E-Cdk2 (5). Many other phosphorylation substrates of cyclin A- and/or cyclin E-dependent kinases, including pRB family members p107 and p130 (6-10), E2F transcription factor family members E2F1, E2F2, and E2F3 (11-15), single-stranded DNA binding protein HSSB-p34/RPA (16, 17), NPAT (18), and the newly identified cyclin D-Cdk4/6 substrate DMP1 (19), all stably interact with the kinases. NPAT and DMP1 were in fact identified through their physical interaction with the kinases. Stable interaction with Cdks is also used by the p21Cip1/p27Kip1 family of kinase inhibitors, which themselves can be phosphorylated by the bound kinases (20-23). Stable physical interaction is not only important for recognition of substrates that directly contact the kinase, but also plays important roles in the phosphorylation of substrates that do not come into direct contact with the kinase but are in multimeric complexes with it. In this case, other proteins serve as adaptors to provide docking sites for both the kinase and the substrate. This situation is perhaps best demonstrated by the cyclin A-Cdk2/p107/E2F4-DP1 complex and the cyclin A-Cdk2/E2F1-DP1 complex. The conserved interacting sequences in p107 (6) and E2F1 (12) mediate interaction with cyclin A-Cdk2,
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which can then phosphorylate other proteins in the complexes. For example, cyclin A-Cdk2 phosphorylates the DP1 that is indirectly connected to it in the cyclin A-Cdk2/E2F1-DP1 multimeric complex (12, 13), and this phosphorylation is important for proper cell cycle regulation (11). Although stable physical interaction is clearly important for target determination, the relationship between the stability of interaction and the efficiency of phosphorylation remains to be tested. It is widely assumed that a more efficient interaction leads to a greater extent of phosphorylation. However, it is difficult to determine this relationship by comparing various kinase-substrate pairs since different phosphorylation sites on different substrates can contribute to the efficiency of phosphorylation. In this report, we studied the relationship between stability of interaction and efficiency of phosphorylation by examining the phosphorylation of the same cell cycle regulatory protein (DP1) in different cyclin A-Cdk2 containing complexes. We chose DP1 because it is present in several cyclin A-Cdk2 containing complexes but does not interact directly with cyclin A as described above. We also wished to have the DP1 in different complexes interact with similar adaptor proteins, and wanted to be able to recover the different complexes with the same antibody. It was shown in vitro that cyclin A-mediated phosphorylation and regulation could be transferred to E2F4 via the fusion of the E2F1 cyclin A binding domain to the N-terminus of E2F4 (the E2F41A protein) (13). Therefore, we also studied the cyclin A-Cdk2/ E2F41A-DP1 complex because this complex and the cyclin A-Cdk2/p107/E2F4-DP1 complex provide an ideal pair for determining the relationship between the stability of cyclin A interaction and the efficiency of DP1 phosphorylation. In addition, the functional consequences of DP1 phosphorylation by cyclin A-Cdk2 were examined. Finally, since E2F4 is predominantly localized in the cytoplasm while the cyclin A binding domain of E2F1 also serves as a nuclear localization signal (24), the effects of forced nuclear localization of E2F4 were determined. We show that although the adaptor proteins are required to attach cyclin A-Cdk2 to the complexes with DP1, a more stable cyclin A interaction does not translate into a more efficient phosphorylation of DP1 in multimeric complexes. In fact, a weaker interaction led to a more efficient phosphorylation. This phosphorylation results in the functional down-regulation of the transactivation activity of the E2F41A-DP1 heterodimer, and this effect is not caused by the change in the cellular localization of E2F4. Mechanisms other than the stability of protein-protein interaction must exist to determine the efficiency of phosphorylation after the kinase is tethered into the complexes.
MATERIALS AND METHODS Plasmids. Plasmids Rc/CMV-E2F4, Rc/CMV-E2F41A, and Rc/ CMV-E2F41Adel24 were constructed by cloning the HindIII-to-XbaI fragments from the corresponding pcDNA1 plasmids (listed below) into the Rc/CMV vector (Invitrogen). pcDNA1-E2F4 was a gift from Doron Ginsberg and David Livingston (25). pcDNA1-E2F41A and pcDNA1-E2F41Adel24 were constructed by inserting PCR amplified BamHI-to-BglII fragments coding for the cyclin A binding domain of E2F1 or its del24 derivative (13) in-frame into the BamHI site of pcDNA1-E2F4. pcDNA3-HA-E2F-4.NLS (26), pCMV-HA-DP1 (27), pcDNA1-HADP1 and pcDNA1-HA-DP1(DP) (11), pCMV-cyclin A (28), pCMVCdk2 and pCMV-Cdk2DN (29), pCMV-p107 and pCMV-p107L19 (30), and pE2F-TK-Luc (31) have been described previously. Cell culture and transient transfection. Human osteosarcoma (U2OS) cells were grown in 5% CO 2 in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum (Gemini BioProducts) in 10 cm plates unless stated otherwise. Cells were transfected by calcium-phosphate methods (32). The following amounts of plasmid DNA were used in transfections (except for luciferase assays, which are described below): E2Fs: 11 ug; DP1: 11 ug; cyclin A: 10 ug; Cdk2: 5.3 ug; Cdk2DN: 5.3 ug; p107: 10 ug; and p107L19: 10 ug. Empty vector DNA was used at amounts equivalent to the other samples for control. For all assays, cells were harvested 42-48 hours after transfection. Immunoprecipitation and Western blotting. Cells were lysed in the presence of protease inhibitors (33) for 30 minutes on ice and cell debris was pelleted. Supernatants were immunoprecipitated for 60 minutes with the indicated antibodies that had been covalently coupled to protein A sepharose. After washing with ELB, samples were boiled in Laemmli buffer and resolved by SDS-PAGE. For Western blotting, proteins were transferred to Immobilon membrane (Millipore) and probed with the indicated antibodies. Anti-DP1 (WTH 16), anti-E2F4 (WUF 10 and 23), anti-E2F1 (KH 20 and 95) and antip107 (SD4) were provided by Ed Harlow. Anti-cyclin A (H-432) was purchased from Santa Cruz Biotechnology. HRP-conjugated mouse or rabbit IgG was used as secondary antibody (Amersham) and detection was performed by ECL (Amersham). Luciferase assays. Cells were seeded into 6-well plates and transfected with the following: 3 ug of E2F-TK-Luc reporter, 110 ng of E2F expression plasmid, 110 ng of DP1, and either 4.4 ug of vector plasmid or 3.3 ug of cyclin A plus 1.1 ug of Cdk2. For the samples lacking E2F4 and DP1, 220 ng of additional vector plasmid was substituted. Separate transfections were done to generate duplicates. Luciferase assays were performed as previously described (34), using D-luciferin substrate (Molecular Probes). Light output was measured for 10 seconds in a Berthold AutoLumat LB 953.
RESULTS We chose DP1 in multimeric complexes to study the relationship between the stability of cyclin A-Cdk2 interaction and the efficiency of phosphorylation. To assemble the various multimeric protein complexes pictured in Figure 1A in vivo, we overexpressed each of the components in U2OS cells through transient transfection. The degree of cyclin A association with each complex was determined by its co-immunoprecipitation with the E2F subunit as shown in Figure 1B. Immunoprecipitation of endogenous and overproduced wild type E2F4 brought down a similar, detectable amount of cyclin A. Since E2F4 does not contain cyclin A binding sequences found in E2F1 and therefore does
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FIG. 1. (A) E2F1 can bind directly to cyclin A, while E2F4 cannot. E2F4 can indirectly associate with cyclin A when it is tethered to it by the p107 protein. Fusion of E2F1’s cyclin A-binding domain onto the N-terminus of E2F4 enables the resulting fusion protein (E2F41A) to bind directly to cyclin A. Mutants E2F1del24 and E2F41Adel24 (lacking 24 amino acids within the cyclin A binding domain) were studied in parallel. (B) U2OS cells were transiently transfected with the indicated expression plasmids and whole cell lysates subjected to immunoprecipitation and Western blotting with the indicated antibodies to determine the levels of association with cyclin A.
not bind cyclin A directly (13), this association is largely through the endogenous p107 protein since immunodepletion of endogenous p107 before E2F4 immunoprecipitation greatly reduced cyclin A association with E2F4 (data not shown). In the same assay, E2F41A associated with about three to five fold more cyclin A than wild type E2F4, demonstrating the contribution of the E2F1 cyclin A binding domain. The
level of cyclin A association with E2F41A was similar to the level of association between E2F1 and cyclin A after the overexpression of E2F1, although in this case a different antibody was used for immunoprecipitation. The corresponding control mutants E2F1del24 and E2F41Adel24, which lacked 24 essential residues within the cyclin A binding domain, bound much less cyclin A.
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FIG. 2. After transient transfection of the indicated expression plasmids into U2OS cells, whole cell lysates were immunoprecipitated and Western blotted with the indicated antibodies to examine the E2F-DP1 associations.
Formation of the cyclin A-Cdk2/p107/E2F4-DP1 complex through co-expression of p107 enabled E2F4 to associate with a much greater amount of cyclin A than E2F41A (Figure 1B). The p107-dependent nature of this was verified by overexpressing L19, a p107 mutant able to bind cyclin A but unable to bind E2F (30), which restored the level of cyclin A association back to that of E2F4 with no added p107. Immunoprecipitation with an anti-p107 antibody brought down large amounts of cyclin A in both p107 and L19 transfected cells. The levels of the overexpressed E2F4, E2F1, and cyclin A proteins were comparable to themselves in all samples (data not shown). Thus, the association of cyclin A with the E2F1-DP1 complex or the E2F41A-DP1 complex was of much lower stability than its association with the p107/E2F4-DP1 complex. The dramatic difference seen in the latter two complexes was directly comparable since the two complexes were recovered through immunoprecipitation with the same antibody. Since phosphorylation of DP1 could be affected by its interaction with E2F1 or E2F4 in these complexes, we recovered DP1 through its co-immunoprecipitation with E2F. As shown in Figure 2, E2F4, E2F41A, and E2F41Adel24 in various E2F4-DP1 complexes and E2F4 in p107/E2F4-DP1 complexes all associated with DP1 to a similar extent as determined by immunoprecipitation and Western blotting. Immunoprecipitation with an anti-E2F1 antibody also brought down comparable levels of DP1 from the E2F1-DP1 complexes. While DP1 was present in all of these complexes, a clear difference in the electromobility patterns of the associated DP1 proteins was observed. DP1 associated with E2F1 and with E2F41A displayed an upward smear which was not noticeable when it was associated with E2F1del24, E2F4, E2F41Adel24, or E2F4 in the p107-containing complexes. To determine whether this type of migration pattern was due to hyper-phosphorylation, we treated the antiE2F4 immunoprecipitates with lambda protein phos-
phatase. The phosphatase treatment compressed the upward smear of E2F41A-associated DP1 (data not shown). We also performed this experiment in the presence of phosphatase inhibitors, which restored the banding pattern of the E2F41A-associated DP1 to that seen when only a mock phosphatase treatment was done (data not shown). Thus, the upward smear pattern of E2F41A-associated DP1 was due to hyperphosphorylation. To test whether the DP1 hyper-phosphorylation was dependent on Cdk2, we utilized a kinase-inactive mutant of this protein, referred to as Cdk2DN (29). When E2F41A and DP1 were co-expressed with Cdk2DN, the smeared banding pattern of the E2F41Aassociated DP1 protein was not observed, indicating that Cdk2 activity was responsible for the E2F41Amediated phosphorylation of DP1 (Figure 3A). The two forms of Cdk2 proteins complexed with the corresponding E2F4 proteins to a similar extent (data not shown). Consistent with the increased binding of cyclin A shown in Figure 1B, E2F41A associated with a greater amount of Cdk2 (both WT and DN) than E2F4 did (data not shown). Endogenous cyclin A-Cdk2 proteins produced an intermediate extent of E2F41Aassociated DP1 phosphorylation, between those resulting from overexpression of cyclin A-Cdk2 and of cyclin A-Cdk2DN (data not shown). Recently, the N-terminus of E2F1 has been shown to be responsible for the nuclear localization of E2F1, in addition to cyclin A binding (24). While E2F1-DP1 is nuclear, overexpressed E2F4-DP1 is localized in the cytoplasm (26, 35, 36). Consistent with these reports, immunofluorescence studies with an anti-E2F4 antibody indicated that E2F4 was cytoplasmic, but E2F41A was nuclear (data not shown). E2F1 is more potent in transactivation and promotion of S phase entry than E2F4, and it was shown that all of the functional differences between E2F1 and E2F4 were attributable to the different cellular localization of these two E2Fs. By the generation of chimeras, it was
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FIG. 3. U2OS cells were transiently transfected with the indicated expression plasmids, including a dominant negative version of Cdk2 (Cdk2DN) and a nuclear localized mutant of E2F4 (E2F4.NLS). Whole cell lysates were immunoprecipitated and analyzed by Western blotting with the indicated antibodies to (A) assess the role of Cdk2 activity in mediating DP1 hyper-phosphorylation, and to (B) determine the role of nuclear localization of E2F4 in mediating association with cyclin A and hyper-phosphorylation of DP1.
shown that the N-terminus of E2F1 (residues 1 to 126, containing the E2F1 cyclin A binding domain), or even the SV40 T antigen nuclear localization sequence, was sufficient to transport E2F4 into the nucleus and stimulate E2F4 activity in transactivation and S phase promotion assays (24). We therefore determined whether nuclear localization of E2F41A was responsible for its effects on DP1 phosphorylation. For this purpose, an E2F4 protein fused with the nuclear localization sequence of SV40 T antigen (E2F4.NLS, (26)) was compared with E2F41A. E2F4.NLS was co-transfected with DP1 and cyclin A-Cdk2 in parallel with E2F4 and E2F41A. Cell extracts were immunoprecipitated with an anti-E2F4 antibody, and the interaction with cyclin A and the phosphorylation status of co-precipitated DP1 were determined as before. Figure 3B shows that E2F4.NLS overexpression did not increase the interaction with cyclin A over that of E2F4, in spite of the similarity between the SV40 T antigen NLS (PKKKRKV) and the E2F1 cyclin A binding domain (PVKRRLDL) (12, 37). Overexpression of E2F4.NLS also did not promote DP1
hyper-phosphorylation like E2F41A, as indicated by the lack of the upward smear of the associated DP1 (Figure 3B). Protein expression levels of E2F4.NLS were comparable with that of E2F41A (data not shown). Therefore, simple nuclear localization without cyclin A interaction is not sufficient for efficient DP1 phosphorylation. This result is in fact consistent with our results obtained from the cyclin A-Cdk2/p107/ E2F4-DP1 complex. Although E2F4 is nuclear when associated with p107 ((26, 35) and data not shown), the formation of cyclin A-Cdk2/p107/E2F4-DP1 complexes did not result in hyper-phosphorylation of this E2F4associated DP1 (Figure 2). We investigated the transactivation potentials of E2F4-DP1 and E2F41A-DP1 in the presence and absence of exogenous cyclin A-Cdk2. After transient transfection of an E2F site-containing luciferase reporter plasmid and the indicated expression constructs, whole cell extracts were assayed for luciferase activity. E2F4-DP1, E2F41A-DP1, and E2F41Adel24DP1 all displayed similar transactivation activities (Figure 4A) under the conditions used in these exper-
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FIG. 4. (A) U2OS cells were transiently transfected with the indicated expression plasmids, along with an E2F-luciferase reporter plasmid. All transfections were performed in duplicate. Whole cell extracts were analyzed for luciferase activity over a 10 sec. time period. The values depicted in the graphs are the averages of duplicates, and the error bars show the deviation from the average value. The luciferase activity of E2F4-DP1 in each experiment was arbitrarily set to a value of 1.0, and was consistently 8-10 fold higher than the activity derived from reporter only-transfected extracts. The data shown are from representative experiments out of four. (B) After transient transfection of the indicated expression plasmids into U2OS cells, whole cell lysates were analyzed by immunoprecipitation and Western blotting with the indicated antibodies to assess the extent of phosphorylation of the E2F4-associated DP1 and DP1(DP) proteins. DP1(DP) contains a total of five serine/threonine to alanine mutations, and is recognized by the monoclonal antibody raised against DP1. Luciferase assays were performed as described in part (A). Shown are the data from a representative experiment out of three.
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iments. However, co-expression of cyclin A-Cdk2 revealed significant differences among their regulation. The transactivation activities of E2F4-DP1 and E2F41Adel24-DP1 were significantly stimulated by the inclusion of cyclin A-Cdk2 (about 3.5 fold each), likely due, at least in part, to the hyper-phosphorylation of cellular pocket proteins by the overexpressed cyclin A-Cdk2 and the consequent release of E2F from pocket protein-mediated repression (38). Indeed, expression of cyclin A-Cdk2 alone stimulated the E2F reporter activity (about 2 fold) over the vector control (data not shown). However, the stimulatory effect of cyclin A-Cdk2 on E2F4-DP1 was not observed on E2F41A-DP1, indicating that the tethering of cyclin A-Cdk2 onto E2F4 resulted in negative regulation. It was shown that the transactivation activity of E2F1 was down-regulated by cyclin A-Cdk2, dependent upon the stable interaction between E2F1 and cyclin A (12). Our results show that direct binding of cyclin A-Cdk2 to E2F41A can serve to down-regulate the transactivation activity of E2F4 as well. Nuclear localization of E2F4 through the SV40 T antigen nuclear localization sequence (the E2F4.NLS protein) again was not sufficient to produce the effects observed from E2F41A (Figure 4A). The activity of E2F4.NLS-DP1 was higher than that of E2F4-DP1 in the absence of transfected cyclin A-Cdk2. In addition, co-transfected cyclin A-Cdk2 strongly stimulated the transactivation activity of E2F4.NLS-DP1 (about 4 fold), further demonstrating the functional differences between E2F41A and E2F4.NLS. To test whether hyper-phosphorylation of DP1 in association with E2F41A was responsible for the observed functional down-regulation, we tested DP1(DP) in these assays. DP1(DP) is a mouse DP1 with Ala subtitutions in five potential Ser and Thr phosphorylation sites. It displayed diminished ability to be phosphorylated in in vitro kinase assays and produced S phase accumulation in cells infected with DP1(DP)-expressing viruses (11). Although this mutant still retained one potential phosphorylation site, it was clear from Figure 4B that it could not be hyper-phosphorylated by cyclin A-Cdk2 in association with E2F41A in vivo. In the transactivation assay, the activity of E2F41A-DP1(DP) was not significantly down-regulated by cyclin A-Cdk2, compared with DP1 in the same experiment (Figure 4B). Since the original DP1(DP) construct was based on vector pcDNA1, we also used pcDNA1-DP1 in these experiments, which consistently showed less activation by co-transfected cyclin A-Cdk2. These results confirm that the observed functional downregulation of E2F41A-DP1 was through the hyperphosphorylation of DP1 by the weakly associated cyclin A-Cdk2.
DISCUSSION In addition to the short consensus sequence flanking the phosphoacceptor site (S/T-P-polar residue-basic residue), stable protein-protein interaction has been found to be an important mechanism for substrate recognition by cyclin-dependent kinases (Cdks). The interaction can exist between the kinase and its substrate, or between the kinase and an adaptor molecule that in turn provides a docking site for the substrate. These two kinds of enzyme-substrate interactions are also important for other kinase-mediated regulations, such as JNK regulation of the jun family in response to extracellular signals (39). The presence in the cell of numerous multimeric complexes involving Cdks strongly indicates that indirect interaction through adaptor molecules is an important regulatory mechanism. For multimeric complexes, the nature and organization of adaptor proteins could be another important factor in determining the specificity and efficiency of regulation by the Cdks, in addition to stable interaction. The relative contributions of these two factors to the regulation have not been characterized, although it is usually assumed that stronger interactions are better for regulation since they can more efficiently increase the local concentration of the kinases. In this study, we have tested this assumption by comparing the phosphorylation of DP1 in the cyclin A-Cdk2/p107/ E2F4-DP1 complex and in the cyclin A-Cdk2/E2F41ADP1 complex. The results demonstrate that, while DP1 in the E2F4-DP1 dimer is not hyper-phosphorylated due to the lack of stable physical interaction between E2F4 and cyclin A, very stable cyclin A association through p107 also does not result in DP1 hyper-phosphorylation. On the other hand, weak association of cyclin A with E2F1, or with E2F41A, leads to efficient phosphorylation of DP1. Therefore, the stability of the interaction of cyclin A-Cdk2 with the complex does not dictate the phosphorylation efficiency of a substrate in the complex. The nature and structural organization of the adaptor proteins is important in determining the actual phosphorylation efficiency. It is conceivable that while certain subunit arrangement in the multimeric complex will help to bring the substrate closer to the kinase, other arrangement will actually restrict the access of the substrate to the kinase, even when the interaction of cyclin-Cdk with the complex is very stable. This might be the case with the cyclin A-Cdk2/ p107/E2F4-DP1 complex, in which p107 may only direct cyclin A-Cdk2 phosphorylation towards itself (7, 21, 40). Future structural studies of these multimeric complexes will provide physical bases for the targeting specificity in multimeric complexes. While the identification of consensus cyclininteracting sequences in many unrelated proteins pro-
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vides a structural explanation for enzyme-substrate interaction, at the same time it adds complexity to the issue of enzyme-substrate specificity. If most of the substrates use similar sequences to interact with cyclin-dependent kinases, how then is the specificity determined among these substrates? Use of different adaptor proteins can provide the specificity for substrates that indirectly interact with the kinase through the adaptors. This is perhaps best demonstrated by the F-box proteins that determine the target specificity of ubiquitin-mediated protein degradation in proteasomes. Different F-box proteins tether the same SCF ubiquitin-ligase complex to different target proteins (41). Our findings indicate the presence of a further level of specificity determination. After the Cdks are tethered to the complexes, the phosphorylation efficiency of a substrate in the complexes still needs to be determined by the nature of its adaptor proteins and the arrangement of the subunits in the complexes. Our results also reveal an important phenomenon regarding the regulation of E2F4-DP1 by the N-terminus of E2F1. A prominent difference among the E2F family members is their relationship with the cyclin A-dependent kinases. E2F1 contains a dedicated domain for direct association with cyclin A while E2F4 does not. Interestingly, this domain also serves as a nuclear localization sequence for E2F1, and the simple nuclear localization of E2F4 was shown to account for the functional differences between E2F1 and E2F4 (24). Our results show that the hyper-phosphorylation of E2F41A-associated DP1 and the down-regulation of the heterodimer’s transactivation activity are dependent on binding to cyclin A-Cdk2, not just on nuclear localization. Currently, studies are in progress to determine the effects on the cell cycle and on target gene expression by this kind of mis-regulation of E2F4. ACKNOWLEDGMENTS We thank Doron Ginsberg, Wilhelm Krek and David Livingston; Sander van den Heuvel and Ed Harlow; Philip Hinds and Robert Weinberg; and Richard Pestell for various reagents used in this study, and Anthony Karnezis for critical reading of the manuscript. We also thank the Pestell lab for assistance with luciferase assays. This work was supported by an American Cancer Society Research Project Grant 97-125-01. The Albert Einstein Cancer Center core support is also acknowledged. P.G. was supported by an NIH Cell and Molecular Biology Training Grant and L.Z. is a Leukemia Society of America Scholar.
REFERENCES 1. Morgan, D. D. (1997) Annu. Rev. Cell Dev. Biol. 13, 261–291. 2. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499 –511. 3. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J., and Livingston, D. M. (1993) Cell 73, 487– 497. 4. Ludlow, J. W., and Skuse, G. R. (1995) Virus Research 35, 113– 121.
5. Kelly, B. L., Wolfe, K. G., and Roberts, J. M. (1998) Proc. Natl. Acad. Aci. USA 95, 2535–2540. 6. Ewen, M., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85– 87. 7. Faha, B., Ewen, M., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87–90. 8. Lees, E., Faha, B. F., Dulic, V., Reed, S. I., and Harlow, E. (1992) Genes Dev. 6, 1874 –1885. 9. Li, Y., Graham, C., Lacy, S., Duncan, A. M. V., and Whyte, P. (1993) Genes Dev. 7, 2366 –2377. 10. Peeper, D. S., Parker, L. L., Ewen, M. E., Toebes, M., Hall, F. L., Xu, M., Zantema, A., van der Eb, A. J., and Piwnica-Worms, H. (1993) EMBO J. 12, 1947–54. 11. Krek, W., Xu, G., and Livingston, D. M. (1995) Cell 83, 1149 – 1158. 12. Krek, W., Ewen, M. E., Shirodkar, S. Z., Arany, Z., Kaelin, W. G., and Livingston, D.M. (1994) Cell 78, 161–172. 13. Dynlacht, B. D., Moberg, K., Lees, J. A., Harlow, E., and Zhu, L. (1997) Mol. Cell. Biol. 17, 3867–3875. 14. Dynlacht, B. D., Flores, O., Lees, J. A., and Harlow, E. (1994) Genes Dev. 8, 1772–1786. 15. Xu, M., Sheppard, K.-A., Peng, C.-Y., Yee, A. S., and PiwnicaWorms, H. (1994) Mol. Cell. Biol. 14, 8420 – 8431. 16. Dutta, A., and Stillman, B. (1992) EMBO J. 11, 2189 –2199. 17. Gibbs, E., Pan, Z. Q., Niu, H., and Hurwitz, J. (1996) J. Biol. Chem. 271, 22847–22854. 18. Zhao, J., Dynlacht, B., Imai, T., Hori, T., and Harlow, E. (1998) Genes Dev. 12, 456 – 461. 19. Hirai, H., and Sherr, C. J. (1996) Mol. Cell. Biol. 16, 6457– 6467. 20. Zhang, H., Hannon, G. J., and Beach, D. (1994) Genes Dev. 8, 1750 –1758. 21. Zhu, L., Harlow, E., and Dynlacht, B. D. (1995b) Genes Dev. 9, 1740 –1752. 22. Blain, S. W., Montalvo, E., and Massague, J. (1996) J. Biol. Chem. 272, 25863–25872. 23. Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M., and Clurman, B. E. (1997) Genes Dev. 11, 1464 –1478. 24. Muller, H., Moroni, M. C., Vigo, E., Petersen, B. O., Bartek, J., and Helin, K. (1997) Mol. Cell. Biol. 17, 5508 –5520. 25. Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z.-X., Xu, G., Wydner, K. L., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 2665–2679. 26. Lindeman, G. J., Gaubatz, S., Livingston, D. M., and Ginsberg, D. (1997) Proc. Natl. Acad. Sci. USA 94, 5095–5100. 27. Helin, K., Wu, C., Fattaey, A., Lees, J., Dynlacht, B., Ngwu, C., and Harlow, E. (1993) Genes Dev. 7, 1850 –1861. 28. Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Cell 70, 993–1006. 29. van den Heuvel, S., and Harlow, E. (1993) Science 262, 2050 – 2054. 30. Zhu, L., Enders, G., Lees, J. A., Beijersbergen, R. L., Bernards, R., and Harlow, E. (1995a) EMBO J. 14, 1904 –1913. 31. Pestell, R. G., Albanese, C., Lee, R. J., Watanabe, G., E., M., Johnson, J., and Jameson, J. L. (1996) Cell Growth Differ. 7, 1337–1344. 32. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1990) Current Protocols in Molecular Biology. Wiley Interscience, New York. 33. Jiang, H., Chou, H. S., and Zhu, L. (1998) Mol. Cell. Biol. 18, 5284 –5290. 34. Watanabe, G., Albanese, C., Lee, R. J., Reutens, A., Vairo, G.,
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Henglein, R., and Pestell, R. G. (1998) Mol. Cell. Biol. 18, 3212– 3222. 35. Magae, J., Wu, C. L., Illenye, S., Harlow, E., and Heintz, N. H. (1996) J. Cell Sci. 107, 1717–1726. 36. Verona, R., Moberg, K., Estes, S., Starz, M., Vernon, J. P., and Lees, J. A. (1997) Mol. Cell. Biol. 17, 7268 –7282. 37. Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nallin, C. M., and Kaelin, J. W. G. (1996) Mol. Cell. Biol. 16, 6623– 6633.
38. Bremner, R., Cohen, B. L., Sopta, M., Hamel, P. A., Ingles, C. J., Gallie, B. L., and Phillips, R. A. (1995) Mol. Cell. Biol. 15, 3256 –3265. 39. Kallunki, T., Deng, T., Hibi, M., and Karin, M. (1996) Cell 87, 929 –939. 40. Woo, M. S., Sanchez, I., and Dynlacht, B. D. (1997) Mol. Cell. Biol. 17, 3566 –3579. 41. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J., and Harper, J. W. (1997) Cell 91, 209 –219.
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