Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Abstract

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.

FIG. 1.

A cluster of four phosphorylation sites destabilizes Cln2. (A) Cln2 protein coding region. The seven Cdc28 phosphorylation sites are numbered (1, T311; 2, T381; 3, S396; 4, T405; 5, S427; 6, T430; 7, S518). The PEST domain is indicated as hatched box. Abbreviations: X, amino acid substitution to alanine; D, the region in mutant M46 harboring the four relevant substitutions that render Cln2 stable. (B) Stability of wild-type and mutant Cln2. Wild-type Cln21 and mutant Cln2 tagged with an HA epitope were pregrown under noninducing conditions (2% raffinose). GAL1-CLN2 or its derivatives were expressed for 45 min by addition of 2% galactose and then repressed by addition of 2% glucose. Extracts were prepared from samples taken at the indicated times and analyzed by immunoblotting using anti-HA antibodies. Equal loading of samples was verified by incubating the blots with anti-Cdc28 antibodies. (C) Graph representing degradation rates from at least two independent experiments. Values were normalized to Cdc28 as internal loading control and to a dilution series (see Materials and Methods).

FIG. 2.

Grr1 interacts with phosphorylated Cln2 but not with hypophosphorylated Cln2^4T3S or Cln2^M46. Lysates from strains expressing either untagged GRR1 (lanes 1 and 4) or chromosomally 6His-tagged GRR1 (lanes 2, 3, 5, 6, and 7 to 10) from its endogenous promoter and expressing wild-type (wt) CLN2HA (lanes 1, 2, 4, 5, 7, and 9), CLN^24T3S-HA (lanes 3 and 6), or CLN2^M46-HA (lanes 8 and 10) from the GAL1 promoter were immunoprecipitated (IP) with a monoclonal anti-HA antibody (lanes 4 to 6, 9, and 10). Immunoprecipitates were immunoblotted with anti-His antibody to detect Grr1-6His, with 12CA5 antibody to detect Cln2HA, or with anti-Cdc28 antibody. IgG, immunoglobulin G.

FIG. 3.

The Cln2-2C and -PD domains confer phosphorylation-dependent instability upon a stable ΔN-Sic1 reporter protein. (A) Domains of Cln2 fused to reporter proteins. The top bar shows the CLN2 C terminus containing the PEST domain (open box) and six Cdc28 phosphorylation sites. Below are the Cln2 domains that were fused to the reporter proteins; mutations of serine or threonine residues to alanines are marked (X). (B) Wild-type Sic-HA, stabilized Sic^mut-HA harboring mutations in four phosphorylation sites, or ΔN-Sic1-HA fused to the indicated Cln2 domains was subjected to GAL1 promoter shutoff experiments as described in Fig. 1. Extracts from samples taken at the indicated time points after repression of the GAL1 promoter were analyzed by immunoblotting using anti-HA antibodies. As a loading control, anti-Cdc28 antibodies were used. (C) Graph representing the degradation rates of the various fusions.

FIG. 4.

The Cln2-2C and -PD domain fusions are phosphorylated and ubiquitinated in vivo. (A) Cln2-ΔN-Sic1-HA fusion proteins were captured on anti-HA beads and incubated for 30 min with CIP in the presence (+) or absence (−) of phosphatase inhibitors, followed by immunoblotting using anti-HA antibodies. The encircled “P” designates phosphorylated species. (B) Extracts prepared from cells expressing the indicated Cln2-ΔN-Sic1-6His-HA fusions and 6His-Myc-tagged K48R, G76A mutant ubiquitin (6His-MYC-UBI-RA) were chromatographed over Ni-NTA beads in a buffer containing 6 M guanidine hydrochloride. Bound proteins and ubiquitin conjugates were analyzed by immunoblotting using anti-HA antibodies.

FIG. 5.

Grr1 interacts with Cln2-2C, D, and PD fused to ΔN-Sic1-HA-6His. (A) Lysates from strains expressing either untagged GRR1 (lanes 1 and 8) or chromosomally 6His-tagged GRR1 (lanes 2, 3, 5, 6, and 7 to 10) from its endogenous promoter and those expressing wild-type CLN2HA (lanes 7 and 14), or the indicated Cln2 domain fusions to ΔN-Sic1-HA-6His from the GAL1 promoter, were immunoprecipitated (IP) with a monoclonal anti-HA antibody (α-HA) (lanes 8 to 14). Immunoprecipitates were immunoblotted with anti-His antibody (α-His) to detect Grr1-6His or with anti-HA antibody to detect Cln2HA. (B) Cdc4 fails to interact with 2C-ΔN-Sic1-HA-6His. Lysates from strains expressing chromosomally HA-tagged CDC4 from its endogenous promoter and expressing 2C-ΔN-Sic1-HA-6His (lanes 1 to 3) or Sic1-HA-6His (lanes 4 to 6) from the GAL1 promoter were incubated with Ni²⁺-NTA beads. Extracts (L), flowthrough (F), and bound proteins (B) were analyzed by immunoblotting with anti-HA antibody.

FIG. 6.

(A) Cdc28 binds to Cln2-ΔN-Sic1 fusions. Strains expressing the indicated fusions (lanes 4 to 18) or a mutant Cln2Δxs lacking its cyclin box were subjected to coimmunoprecipitation and immunoblotting as described in the legend to Fig. 5, except that anti-Cdc28 antibody (α-Cdc28) was used to detect bound Cdc28. Abbreviations: E, extract; F, flowthrough; B, bound; α-HA, anti-HA antibody. (B) Deletion of the Sic1 C terminus results in loss of Clb-Cdc28 binding. Strains expressing 2C-ΔN-Sic1 (lanes 4 and 8) or Cln2 domains fused to ΔN-Sic1 lacking its C terminus (lanes 1 to 3 and 5 to 7) were subjected to coimmunoprecipitation (IP) and immunoblotting as described in the legend to Fig. 5, except that anti-Cdc28 antibody was used to detect bound Cdc28. (C) Deletion of the Sic1 C terminus does not affect the stability of 2C-ΔN-Sic1. 2C-ΔN-Sic1-HA and 2C-ΔN-Sic1-HA with a C-terminal deletion of the Clb/Cdc28 binding site were subjected to GAL1 promoter shutoff experiments as described in the legend to Fig. 1. Extracts from samples taken at the indicated time points after repression of the GAL1 promoter were analyzed by immunoblotting using anti-HA antibodies. As a loading control, anti-Cdc28 antibodies were used.

FIG. 7.

Release of Cdc28 from phosphorylated Cln2 does not affect binding to Grr1. Extracts from strains expressing Cln2HA (lanes 1 to 4 and 9 to 12) or not expressing Cln2HA (lanes 5 to 8) were incubated with anti-HA beads for 1 h and washed two times under low-stringency (lysis buffer; lanes 1, 3, 5, 7, 9, and 11) or high-stringency (lysis buffer + 1% SDS) conditions, followed by incubation in extracts from insects cells expressing 6His-Grr1 (lanes 1, 2, 5, and 6), mock extracts (lanes 3, 4, 7, and 8), Ni-NTA-purified 6His-Grr1 (lanes 9, 10), or mock-purified extracts (lanes 11 and 12) for 1 h at 4°C. Beads were washed and boiled in 2× SDS sample buffer, and bound proteins were analyzed by immunoblotting using anti-His antibody to detect 6His-Grr1, anti-HA antibody to detect Cln2HA, and anti-Cdc28 antibody. IP, immunoprecipitation.

FIG. 8.

Cln2-ΔN-Sic1 fusions are degraded by SCF^Grr1. (A) Cln2 fusions alleviate the lethality of stabilized Sic1. Strains expressing Sic1; stabilized Sic1^4A (Sic1^m); empty vector (vec); Cln2; or fusions of stabilized Sic1 (ΔN-Sic1) with 2C (2C), mutant 2C^M46 (2C^m), and PD (PD)—all under control of the GAL1 promoter—were replica plated onto glucose- and galactose-containing plates and incubated at 30°C for 3 days. (B) Grr1 is essential in strains expressing Cln2-ΔN-Sic1 fusions. Wild type (wt) strains or strains with Grr1 deleted (grr1Δ) and expressing 2C-ΔN-Sic1 (2C), or PD-ΔN-Sic1 (PD) under control of the GAL1 promoter, were replica plated onto glucose- and galactose-containing plates and incubated at 30°C for 3 days. (C) Degradation of 2C and PD fusions to ΔN-Sic1 depends on functional SCF^Grr1. 2C-ΔN-Sic1-HA or Sic1-HA was expressed in a wt strain, in a grr1Δ strain, or in temperature-sensitive strains (cdc53ts and cdc4ts) grown at 37°C and subjected to GAL1 promoter shutoff experiments as described in the legend to Fig. 1. Extracts from samples taken at the indicated time points after repression of the GAL1 promoter were analyzed by immunoblotting using anti-HA antibodies. (D) Graph representing the degradation rate of 2C-ΔN-Sic1 in the indicated strains.

FIG. 9.

Model of phosphorylation-induced binding of SCF^Grr1 to Cln2. For an explanation, see text. Abbreviations: CB, cyclin box; P, Cln2 PEST domain; D, Cln2 D domain; F, Grr1 F box; LRR, Grr1 LRR domain; 34, Cdc34; 53, Cdc53.