The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation

Abstract

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

Fig. 1.

The Cul1 Cycle. A, Cul1 complexed with FBP-Skp1 and covalently modified with Nedd8 (N8) represents an active SCF. B and C, Inactivation of SCF initiates when CSN binds active SCF and deconjugates Nedd8, returning the RING subunit Rbx1 to its inactive configuration. D, Cand1 dislocates FBP-Skp1 and binds Cul1, forming an inactive Cand1-Cul1 complex. Restoration of an active SCF complex is brought upon by the combined action of Nedd8-conjugating enzymes and FBP-Skp1. Conjugation of Nedd8 causes a major conformational change in Cul1 and E2-recruiting Rbx1. This dynamic cycle of Cul1 is thought to enable a rapid sampling of the steady-state FBP-Skp1 population.

Fig. 2.

Generation of a stable cell line expressing tagged Cul1 upon tetracycline treatment. Flip-In T-REx 293 cells were transfected with pCDNA5/FRT/TO vector containing sequences encoding Cul1^HTBH, and selected with hygromycin (100 μg/ml). Selected cells were induced with the indicated amount of tetracycline for 4 h (Lane 1, no tetracycline; Lane 2, 0.2 μg/ml, Lane 3, 0.5 μg/ml; Lane 4, 1.0 μg/ml). After induction, cells were grown for 24 h and lysed for Western blot analysis.

Fig. 3.

Correlation of the emPAI values and number of Pubmed articles for F-box proteins. Representative F-box proteins that are either intensively studied or found at high levels in Cul1 immuno-preciptates are labeled.

Fig. 4.

Effect of MLN4924 on the neddylation status of Cul1. Flip-In T-REx 293 cells with an integrated CUL1-HTBH gene (Fig. 2) were induced with 1.0 μg/ml tetracycline for 8 h. Cells were grown 24 h before being treated with the indicated amount of MLN4924 for an additional 24 h (Lane 1, no MLN4924; Lane 2, 100 nm; Lane3, 50 nm, Lane 4, 10 nm, Lane 5, 5 nm). Cells were lysed with lysis buffer containing 1 mm 1,10-phenanthroline, a zinc chelator that blocks the deneddylating activity of the COP9 signalosome (CSN). Neddylated species for both Cul1^HTBH and the endogenous Cul1 are indicated with the asterisk marks.

Fig. 5.

Effect of MLN4924 on the availability of free Cand1. Flip-In T-REx 293 cells with an integrated CUL1-HTBH gene were mock-treated or treated with MLN4924 (3 μm MLN4924 for 15 min treatment and 100 nm MLN4924 for 24 h treatment) prior to tetracycline induction. Following this first treatment period, Cul1^HTBH was expressed with 2 μg/ml tetracycline for 2 h, keeping MLN4924 present in the MLN4924-treated samples. Following cell lysis, newly synthesized Cul1^HTBH was affinity purified, and the amount of Cul1-bound Cand1 was analyzed by Western blot.