Fbw7/hCDC4 dimerization regulates its substrate interactions
© Welcker and Clurman; licensee BioMed Central Ltd. 2007
Received: 29 November 2006
Accepted: 13 February 2007
Published: 13 February 2007
The Fbw7 ubiquitin ligase promotes the rapid degradation of several important oncogenes, such as cyclin E, c-Myc, c-Jun, and Notch. The two fission yeast homologs of Fbw7, pop1 and pop2, have previously been shown to dimerize. In this study, we asked whether Fbw7 can also dimerize and how dimerization affects Fbw7 function.
We found that Fbw7 binds efficiently to itself through a domain just upstream of its F-box. We further show that dimerization is essential for the stable interaction of Fbw7 with the cyclin E T380 phospho-degron. Surprisingly, the requirement for dimerization can be suppressed by an additional phosphorylation of this phospho-degron at the +4 position (S384), which creates a binding site with higher affinity for monomeric Fbw7.
Degradation of cyclin E by the Fbw7 pathway can, thus, be conditionally regulated either by Fbw7 dimerization or by hyperphosphorylation of the T380 phospho-degron. Other substrates, which cannot accommodate an extra phosphate in their phospho-degrons, or which don't provide a negatively charged amino acid in the +4 position, may be absolutely dependent on Fbw7 dimerization for their turnover. Our results point to an additional level of regulation for substrate interaction and turnover by Fbw7.
Fbw7 is the mammalian homolog of budding yeast CDC4 and mediates the degradation of several proteins involved in cell growth and division, including cyclin E, c-Myc, c-Jun, Notch, Presenilin, and SREBP [1–10]. Fbw7 recognizes a phospho-epitope, termed CPD (for C dc4 P hospho-D egron), contained within these substrates. Via its F-box, Fbw7 recruits the remainder of an SCF ubiquitin ligase complex, thus promoting substrate ubiquitination and rapid degradation by the proteasome . Mammalian cells contain three Fbw7 isoforms (Fbw7α, Fbw7β, and Fbw7γ) that are produced by alternative splicing and that localize to the nucleoplasm, cytoplasm, and nucleolus, respectively [12–14].
Numerous cancer-associated mutations have been identified within CPDs of Fbw7 substrates that render them insensitive to Fbw7 regulation. Accordingly, the Fbw7 gene is deleted in a large number of tumors. Moreover, many somatic point mutations have been found that eliminate Fbw7's function either by terminating the protein prematurely or disabling its substrate recognition domain, the C-terminal WD40 repeats .
The eight WD40 repeats form a beta-propeller structure creating a phospho-epitope binding pocket that can recognize phosphorylated CPDs . All currently known mammalian CPDs consist of a central phosphorylated threonine immediately followed by proline (pT-P). For substrates like c-Myc, c-Jun, SREBP, and possibly cyclin E, a phosphorylated serine in the +4 position serves as priming phosphate for GSK3 in order to phosphorylate the central threonine. However, beyond simply priming for GSK3, there appear to be additional requirements for this negative charge in the +4 position for substrate turnover by Fbw7.
Alignment of CPDs.
Conservation of the negative charge in the +4 position of CPDs
LLT PPQS GK
IPT PDKE DD
LPT PPLS PS
GET PPLS PI
TLT PPPS DA
PPT PPPE PE
FLT PSPE SP
IYT PFTE DT
LPT PVLE DA
PIS PPPS LK
DVT PESS PE
PLT PTTS PV
IPS PISE RK
NLT PHST NE
PPT PAKT PK
Because the fission yeast Fbw7 homologs, pop1 and pop2, have previously been shown to dimerize , we tested whether Fbw7 can also form dimers and how dimerization affects its functions. We identified a dimerization domain in the region common to all Fbw7 isoforms just upstream of the F-box. Fbw7 mutants that cannot dimerize are fully active toward cyclin E and c-Myc in turnover assays in cells, indicating that dimerization is not strictly required for Fbw7 function. However, we found that stable interactions between dimerization-deficient Fbw7 and cyclin E are largely impaired and that cyclin E turnover by monomeric Fbw7 completely depends on phosphorylated S384 – even by the Fbw7α isoform. Our results link Fbw7 dimerization to the negative charge in a CPD's +4 position and point to an additional level of complexity in substrate turnover by Fbw7.
Identification of an Fbw7 dimerization domain
We then attempted to define the region on Fbw7α necessary for dimerization. A panel of N-terminal truncation mutants was constructed and tested for interaction with full-length Fbw7α as above. This analysis revealed that a region common to all three Fbw7 isoforms (but lacking the isoform-specific N-terminus) was required to bind to full-length Fbw7 (Fig. 1B). However, the M73 truncation mutant (starting at methionine 73 of the common region) was incapable of binding to Fbw7α, suggesting that residues upstream of M73 are essential for dimerization. Substrate interaction is not required for Fbw7 dimerization, since a cancer-associated Fbw7 point mutant that no longer recognizes CPDs, still bound to wild-type Fbw7α (Fig. 1B, last lane).
Fbw7 dimerization in cells
In order to determine whether heterodimerization may be a post-lysis artifact or actually occurs in cells, we analyzed these interactions by immunofluorescence of fixed cells. U2OS cells were grown on coverslips and transfected with each individual Flag-tagged Fbw7 isoform either alone or together with Myc-tagged Fbw7 isoforms in every heterodimeric combination. The localization of Flag-tagged Fbw7 was then assayed by immunofluorescence. As shown in figure 3C, co-expression of the nucleoplasmic Fbw7α protein caused the normally nucleolar Fbw7γ protein to become mislocalized to the nucleoplasm suggesting that they interact in cells, and this was dependent on an intact dimerization domain of Fbw7α. In contrast, co-expression of cytoplasmic Fbw7β did not relocalize Fbw7γ. Consistent with the preference for homodimerization observed in figures 3A and 3B, Fbw7α and Fbw7β were largely unaffected by co-expressing any other isoform (not shown).
Dimerization is not strictly required for Fbw7 function
Conditional requirement for Fbw7 dimers
In addition to its potential priming role for GSK3, S384 phosphorylation participates in the formation of the T380 CPD and is likely to mediate direct contacts with Fbw7 . We therefore directly tested the role of Fbw7 dimerization in binding to cyclin E and the S384 mutants by co-immunoprecipitation. In order to capture this transient interaction we co-expressed a dominant-negative version of Cul1, an essential core component of SCF complexes, in an otherwise identical experiment as in figure 6A. This prevents cyclin E ubiquitination and degradation and allows stable binding of cyclin E to Fbw7 . Lysates were immunoprecipitated against Fbw7α or the dimerization mutant and western blotted for co-immunoprecipitated cyclin E (Fig. 6B). The results confirmed that the stable interaction between the S384A mutant and Fbw7α is largely impaired compared with wild-type cyclin E. This is not simply due to decreased T380 phosphorylation of this mutant (see lysate). In fact, the extent of T380 phosphorylation is comparable between the S384A and S384E mutants, yet only S384E is efficiently bound to Fbw7. This supports the notion that a negative charge in +4 directly contributes to Fbw7 binding.
We have shown that human Fbw7 can form dimers and mapped the dimerization domain to a region just upstream of the F-box. Since this domain is preserved in all Fbw7 splice variants, Fbw7 may, in principle, be able to form both homo- and heterodimers. However, we and others have demonstrated previously that the Fbw7 isoforms localize to distinct subcellular compartments [12, 13, 20]. Due to the absence of suitable antibodies, all Fbw7 localization studies relied on overexpressed tagged Fbw7, and all conclusions were drawn from the expression of a single Fbw7 isoform at a time. Here we show that, at least upon co-overexpression, some isoforms can interact in cells and alter their localization through heterodimerization. This suggests that, in addition to the cis-acting signals that normally direct them to their locations, the stoichiometry of the endogenous Fbw7 isoforms and the extent of heterodimerization may influence their compartmentalization. It is unclear to what extent (hetero)dimerization among the endogenous Fbw7 isoforms occurs. Due to their low abundance, we are currently unable to detect the endogenous Fbw7 proteins and are restricted to studying ectopically expressed Fbw7. One intriguing consequence of heterodimerization is that one Fbw7 isoform may be able to regulate the activity of another isoform by directing it to another cellular address. For instance, the extent of heterodimerization between Fbw7α and Fbw7γ may regulate the amount of nucleolar Fbw7γ and, hence, the activities of nucleolar substrates.
Besides potentially affecting isoform localization, dimerization may directly regulate other functional aspects of Fbw7. For instance, since the dimerization domain is adjacent to the F-box, we initially tested the idea that dimerization may prevent the interaction with Skp1 and, hence, inactivate Fbw7. However, we found no evidence for this, since Skp1 bound indistinguishably to monomeric and dimerized Fbw7 (not shown). Instead, we uncovered a requirement of Fbw7 dimerization for the regulation of certain substrates. Our results establish a link between Fbw7 dimerization and the presence of a negative charge in the +4 position of cyclin E's T380-CPD. The implications of our findings suggest differential modes of action by Fbw7 and sub-divide Fbw7 targets into three groups. First are substrates that always provide the negative charge in +4 via glutamates, aspartates, or constitutive (priming) phosphorylation. This subgroup may not rely on Fbw7 dimerization for their turnover. Second are substrates that sometimes, perhaps conditionally, provide this charge and may switch their CPD affinity in response to environmental cues. Cyclin E is an example of this group and can be degraded independent of Fbw7 dimerization when S384 is phosphorylated, but becomes dependent on S384 phosphorylation for monomeric Fbw7 (see model in figure 7). Also c-Myc may belong to this subgroup, since it has been suggested that S62 became de-phosphorylated after having served as primer for T58 phosphorylation . However, in our assays c-Myc regulation by Fbw7 is not dependent on Fbw7 dimers (Fig. 5D). Finally, some substrates may never provide this charge and therefore are completely dependent on Fbw7 dimerization for their degradation. Such CPDs probably mediate weaker interactions with Fbw7 and may entirely rely on cooperative effects together with other (weak) CPDs. The activities of these substrates may in part be controlled by the state of Fbw7 dimerization and are possibly subjected to an additional level of regulation, if dimerization itself was controlled.
Sic1 requires at least six phosphorylated CPDs for optimal destruction by CDC4, each of which are flawed by the absence of the +4 negative charge, the presence of non-favorable basic amino acids, or both [11, 16]. Therefore, the low-affinity CPDs of Sic1, like cyclin E that is not phosphorylated on S384, may entirely depend on CDC4 dimerization for efficient turnover. Indeed, recent studies have shown that Sic1 degradation requires CDC4 dimerization (Mike Tyers personal communication). Why low affinity CPDs require F-box protein dimerization remains unclear, but perhaps their affinity is so low that the recruitment of several SCF complexes is required for efficient substrate degradation. Dimerized Fbw7 (or CDC4) may simply be twice as efficient in substrate ubiquitination, because two SCF complexes can recruit two E2 ubiquitin ligases, and this may be necessary for the turnover of low-affinity substrates.
Several mechanistic ideas arise from our observations. One possibility is that dimerized Fbw7 binds to two different CPDs concurrently to increase substrate affinity and facilitate more efficient ubiquitination of its substrate. This model would be a plausible explanation to account for the more transient interactions of Fbw7/CDC4 with low-affinity CPDs and is depicted in figure 7D. Alternatively, a dimer may bind to a single CPD via one of the dimer moieties while the other one simply recruits a second E2 enzyme, perhaps for more efficient ubiquitination (Figure 7C). Moreover, a dimerized SCF might elongate the same ubiquitin chain, act on two different chains, or perhaps several dimers elongate one single ubiquitin chain when bound to different CPDs. All of these models may apply in a substrate-specific fashion or even for different CPDs within the same substrate.
Our results suggest that Fbw7's affinity for certain substrates underlies yet another level of regulation that links the negatively charged +4 position of CPDs and Fbw7's ability to form dimers. We propose that weak CPDs that do not contain the + 4 charge must either cooperate with another (weak?) CPD or bring a second SCF to the same low-affinity CPD for efficient substrate destruction via Fbw7 dimerization.
Cell culture, transfections, plasmids
U2OS (human osteosarcoma) and HEK-293A (human embryonic kidney) cell lines were cultured under standard conditions in DMEM containing 10% FCS. For transient transfections experiments, cells were seeded into 6 cm dishes and transfected the next day by Ca-precipitation overnight at 30 to 40% confluence. 24 hrs after washing and replacing the media cells were harvested either for protein extracts or immunostaining. Typically, we express 1/10 of the plasmid amount of pFlag-Fbw7α (0.3 μg) compared to Fbw7β and Fbw7γ (3 μg each). The in vivo Fbw7-driven cyclin E turnover assay was performed as described . All point mutants and deletions were introduced by the Quick Change method (Stratagene) and confirmed by sequencing. The deletions Δ69–72 and Δ74–78 correspond to amino acids EWLK and FQSWS, respectively. Truncation mutants were generated by PCR and sequenced. All amino acid numbers indicate their positions within the common region of Fbw7.
Immunofluorescence, immunoprecipitation, immunoblotting
For immunofluorescence, cells were seeded and transfected on glass coverslips. Slips were fixed with ice-cold Methanol/Acetone (1:1) for 5 min, air-dried and immunostained with primary antibody. After washing with PBS, slips were incubated with secondary FITC-coupled anti-mouse antibody, washed, dried and mounted. For double stainings mouse Flag-tag and rabbit Myc-tag antibodies were mixed and visualized with FITC- and dopamine-coupled secondary antibodies. All protein extracts were made with Tween 20 lysis buffer . Protein expression was determined by standard procedures (SDS-PAGE, western blot, immunoblot). Protein complexes were analyzed by immunoprecipitation. The following primary antibodies were used in this study: anti-Flag for Fbw7 (Sigma, M2), anti-HA for CDK2 and dnCul1 (12CA-5), anti-c-Myc (Santa Cruz, sc-N262), anti-Myc-tag for cyclin E and Fbw7 (9E-10 and Cell Signaling #2278), anti-pT380 for phospho-cyclin E (Santa Cruz, sc-12917-R).
Note added in proof
While this manuscript was under revision a similar study was published by Zhang and Koepp (Mol Cancer Res December 2006).
This work was supported by grants from the National Cancer Institute (NCI) RO1CA84069 and NCI 1 RO1CA102742-01 (B.E.C.) and the Leukemia and Lymphoma Society (M.W.). We like to thank Mike Tyers and Wade Harper for prepublication information, and Steve Reed for 293A cells.
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