The cyclomodulin cycle inhibiting factor (CIF) alters cullin neddylation dynamics

Abstract

The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.

Keywords: CIF; Cullin-RING Ubiquitin Ligases; NEDD8; Signal Transduction; Ubiquitin; Ubiquitin Ligase; Ubiquitin-like Proteins; Ubiquitination; Yeast.

FIGURE 1.

MLN4924 and CIF inhibit CRLs through different mechanisms. A, MLN4924 inhibits the NEDD8-activating enzyme (E1), resulting in general inhibition of the NEDD8 system. This inactivates CRLs (loss of neddylated cullins) and leads to cell death. B, NEDD8 is conjugated to CRLs by the NEDD8 system (E1, E2, and NEDD8 ligase DCN1) and removed by the CSN in a dynamic manner. Exactly how NEDD8 deamidation by CIF inactivates CRLs is unclear, although this is likely to be different from the specific effects of MLN4924 on the NEDD8 E1. Although the CIF product NEDD8 Q40E can be conjugated onto cullins in vitro, it does not appear to support NEDD8-dependent CRL activation (25, 26). Because CIF efficiently deamidates free NEDD8 and NEDD8 in different contexts in vitro (25), CIF could interfere with NEDD8 system function in cells. Finally, NEDD8 recognition and removal by the CSN could be altered by CIF and/or NEDD8 deamidation, changing the extent of activated CRLs.

FIGURE 2.

A yeast strain conditionally dependent on NEDD8 and cullin neddylation dynamics. A, haploid yeast strains that either have RUB1 (encodes yeast NEDD8) or the gene deleted (rub1Δ) were constructed so that the essential functions of the yeast ubiquitin-conjugating enzyme Cdc34 are provided by a counterselectable URA3-marked, plasmid-based copy (MPY101 or MPY143, respectively) and plasmids encoding yeast Cdc34-Myc13, human CDC34-Myc13, or vector control. Five-fold serial dilutions of cells were spotted onto medium in the presence or absence of 5-FOA. Cells on 5-FOA-containing medium harbor the indicated version of CDC34 as the sole source of the essential enzyme. B, lysates from the strains used in A with and without 5-FOA treatment were analyzed by immunoblotting to evaluate the expression of Myc-tagged human and yeast Cdc34, yeast Cdc34, and Cdc28 (loading control). C, the rub1Δ strains from A were provided Rub1 or NEDD8 from expression plasmids using either the Rub1 (RP) or ADH promoter and evaluated as in A. D, lysates from a rub1Δ strain (NEDD8-independent, MPY156) transformed with the same plasmids from C were analyzed by immunoblotting to evaluate the neddylation status of yeast CUL1 (Cdc53) and expression of monomeric NEDD8 and Rub1. A control strain containing endogenous RUB1 is shown for comparison (lane 1). E, the contribution of the CSN on the viability of yeast strains that either express ADH-NEDD8 or vector control was evaluated by comparing strains that are either RRI1⁺ or rri1Δ (MPY143 and MPY202, respectively) in the presence or absence of 5-FOA.

FIGURE 3.

CIF inhibits NEDD8-dependent yeast proliferation. A, the yeast strains from Fig. 2D that express either Rub1 or NEDD8 from the ADH promoter and rely on either human or yeast CDC34 for viability were transformed with plasmids that express galactose-inducible FLAG-tagged CIF (WT), catalytically inactive CIF (C109S), or a vector control. Saturated cultures were serial-diluted and spotted onto medium containing either 2% glucose or galactose to repress or induce CIF expression, respectively. B, the yeast strains from A were diluted to log phase and then induced with either 2% glucose or galactose for 4 h at 30 °C prior to imaging using phase contrast microscopy. Representative fields of cells are shown. C, the plasmids that express galactose-inducible CIF and CIF C109S were transformed into temperature-sensitive strains with mutations in essential CRL pathway components, and cell growth was assessed under the indicated conditions.

FIGURE 4.

CIF and its product NEDD8 Q40E change the extent of cullin neddylation. A, the rub1Δ/pADH-NEDD8 strain (MPY174) coexpressing galactose-inducible CIF (WT or catalytically inactive C109S) was grown to saturation, diluted 1:3 in medium containing either 2% glucose or galactose (GAL) to repress or induce CIF expression, and incubated for 4 h at 30 °C. Lysates from these cells were analyzed by immunoblotting for yCUL1, NEDD8, and FLAG (detects CIF) with Cdc28 as a loading control. B, lysates from a rub1Δ strain (MPY156) expressing NEDD8, NEDD8 Q40E, or a vector control were analyzed by immunoblotting for yCUL1, NEDD8, and Cdc28. C, serial dilutions of the haploid cdc34Δ rub1Δ strain carrying counterselectable yeast Cdc34 and human CDC34 (MPY146) and expressing NEDD8 or NEDD8 Q40E were plated onto medium with 5-FOA to evaluate the effect of Q40E on NEDD8-conditional growth. D, haploid rub1Δ rri1Δ yeast strains (MPY201) coexpressing NEDD8 or NEDD8 Q40E were analyzed as described in B.

FIGURE 5.

An antibody that preferentially recognizes NEDD8 Q40E. A, 2-fold serial dilutions of purified recombinant NEDD8 Q40E or NEDD8 (3.125–200 ng) were analyzed by immunoblot analysis with the indicated antibodies. Graphs of quantitative analyses comparing signal intensity detected using a Licor Odyssey are shown for each (n = 3). Error bars represent S.E. B, yeast lysates from a rub1Δ yeast strain (MPY156) expressing vector control, NEDD8, or NEDD8 Q40E were immunoblotted with the NEDD8 or NEDD8 Q40E antibodies.

FIGURE 6.

Deamidated NEDD8 is removed more efficiently by the CSN than NEDD8. A, haploid rub1Δ yeast strains expressing NEDD8 (MPY174) and galactose-inducible CIF (WT) or catalytically inactive CIF (C109S) were grown to late log phase and induced with galactose. Cell lysates prepared at the times after galactose addition were analyzed by immunoblotting with the indicated antibodies. B, an affinity-purified CSN was added to lysates from rub1Δ rri1Δ strains (MPY201) expressing NEDD8 or NEDD8 Q40E. Samples were analyzed at the indicated times by immunoblotting for yeast CUL1.

FIGURE 7.

NEDD8 deamidation occurs primarily on cullins. Haploid rub1Δ rri1Δ yeast strains expressing NEDD8 (MPY257) and either WT or catalytically inactive (C109S) CIF were analyzed as described in Fig. 6A.

FIGURE 8.

NEDD8 deamidation does not inhibit Ubc12 activation and cullin neddylation. A, lysates from the rub1Δ yeast strain expressing NEDD8 (MPY174) and Myc-tagged Ubc12 with galactose-inducible CIF (WT) or catalytically inactive CIF (C109S) were prepared at the indicated times after 2% galactose induction. Activated Ubc12 (Ubc12-Myc∼NEDD8) was detected by immunoblotting samples prepared under non-reducing conditions (omitting 2-mercaptoethanol from the sample buffer) with anti-Myc antisera and the anti-NEDD8 Q40E antibody. The same samples prepared with 2-mercaptoethanol were also analyzed for Myc-tagged Ubc12, FLAG-tagged CIF, and Cdc28. B, lysates from the rub1Δ yeast strain (MPY156) expressing either NEDD8 or NEDD8 Q40E and Myc-tagged Ubc12 were analyzed under non-reducing and reducing conditions by immunoblotting with the indicated antibodies. C, purified recombinant NEDD8-activating enzyme, UBC12, ATP, and fluorescently labeled NEDD8 were added to yeast lysates prepared from a rub1Δ rri1Δ strain (MPY201) with or without NEDD8 expression. After 30 min at 30 °C, samples were subjected to SDS-PAGE and analyzed for fluorescent NEDD8 by direct gel scanning on a Licor Odyssey. CUL1 neddylation was only detected in lysates lacking NEDD8. Because inactivation of the CSN through rri1Δ leads to complete cullin neddylation in cells containing NEDD8 (e.g. Fig. 4C), lysates from these cells do not have unmodified cullins that could serve as substrates for in vitro neddylation with fluorescent NEDD8. D, in vitro neddylation reactions using either fluorescent NEDD8 (WT) or NEDD8 Q40E were performed using yeast cell lysates from a rub1Δ rri1Δ strain (MPY201) as in C. The reactions were stopped at the indicated times with reducing sample buffer, and NEDD8-modified CUL1 was detected by SDS-PAGE and direct gel scanning using a Licor Odyssey.

FIGURE 9.

Overexpression of DCN1 alleviates CIF-mediated inhibition of neddylation but does not restore NEDD8-dependent cell proliferation. A, the rub1Δ yeast strains expressing NEDD8, galactose-inducible CIF (MPY231), and either ADH-HA-DCN1 (DCN1 overexpression (O/E)) or a vector control (none) were grown to late log phase. Galactose was added to 2%, and samples taken at the indicated times were analyzed by immunoblotting with the indicated antibodies. B, lysates from rub1Δ yeast strains (MPY156) coexpressing either NEDD8 (WT) or NEDD8 Q40E and ADH-HA-DCN1 or a vector control were analyzed by immunoblotting with the indicated antibodies. C, the effect of Dcn1 on NEDD8-dependent cell growth was evaluated in yeast strains expressing galactose-inducible CIF (WT or C109S, top panel) or NEDD8 (Q40E or WT; bottom panel). Serial dilutions were spotted onto glucose- or galactose-containing medium for inducible CIF expression and medium without or with 5-FOA for NEDD8 conditional growth.

FIGURE 10.

CIF alters CRL neddylation dynamics. A, CRLs typically cycle between active and inactive states, depending on cullin neddylation status. The NEDD8 system is responsible for the conjugation of NEDD8 to cullins, whereas the CSN juxtaposes this to provide the requisite homeostasis of activated CRLs. B, CIF deamidates NEDD8 attached to cullins (Fig. 7). This results in direct CRL inactivation because CRLs modified with NEDD8 Q40E are not active in vitro (25, 26) and do not support growth in yeast (Fig. 9). The CSN removes NEDD8 Q40E more efficiently than wild-type NEDD8 from CRLs, resulting in an increase in deneddylated CRLs (Fig. 6). Once removed, NEDD8 Q40E is competent for further rounds of CRL modification (Fig. 8). However, the enhanced deneddylation of deamidated NEDD8 results in reduced steady-state levels of neddylated CRLs (Fig. 4), rendering cells incompetent for vegetative growth (Fig. 3).