Phosphorylation of Ubc9 by Cdk1 Enhances SUMOylation Activity

Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9

Phosphorylation-dependent SUMOylation of the transcription factor NF-E2.

Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation

SUMOylation of p45/p18 heterodimer and p18, but not p45, is enhanced by PKA.

<p>A–B) Various concentrations (8 nM, 15 nM, 30 nM and 60 nM) of PKA were added in SUMOylation reaction mixtures containing His₆-SUMO1, His₆-Ubc9, SAE1/His₆SAE2 (2.25 ng/µl), p45/p18 or p45 alone and ATP, which were then incubated at 37°C for 60 min. The reactions were then boiled in SDS sample buffer and resolved by 11.25% and 12.5% SDS-PAGE, followed by immunoblotting with anti-p45 or p18 antibody. C) GST-p18 or GST-p18K14R was added to the SUMOylation mixture in the increasing concentration (12 nM and 30 nM) of PKA and incubated at 37°C for 60 min. The reactions were subsequently boiled in SDS sample buffer, resolved by 10% SDS-PAGE, and immunoblotted with an anti-GST antibody. D) SUMOylation assay containing His₆-SUMO1, His₆-Ubc9, SAE1/His₆SAE2 (2.25 ng/µl), GST-p18, PKA and ATP were mixed in the presence of various concentration (20 µM, 60 µM and 100 µM ) of PKA inhibitor (Promega) incubated at 37°C for 60 min. The reactions were then boiled in SDS sample buffer and separated by 10% SDS-PAGE, followed by immunoblotting with anti-GST antibody, respectively.</p

Expression and purification of p45/NF-E2 heterodimer and p45.

<p>A) Whole cell lysates of baculovirus co-expressing p45 and p18 were subjected to Mono Q Sepharose anion-exchange chromatography. Proteins were eluted by a NaCl gradient in 50 mM Tris-HCl, pH 8.0 buffer. All isolated fractions (#1–24) were collected by FPLC. The fractions (#11–14) primarily contain the p45/p18 heterodimer. Fractions (#10–17) were subjected to Western blotting with antibody against p45 or p18. B) Eluates containing the p45/p18 heterodimer (fractions #11–14) were subjected to nickel affinity purification and proteins were eluted with 100 mM imidazole. Eluates containing the p45 monomer were similarly purified by nickel affinity column. The purified p45/p18 heterodimer and p45 monomer were resolved by 11.25% and 12.5% SDS-PAGE, respectively, and subsequently immunoblotted with p45 or p18 antibody as indicated.</p

SUMOylation of p45/p18 heterodimer, but not p45, is enhanced by ERK1.

<p>A–B) Various concentrations (9 nM, 18 nM and 35 nM) of ERK1 were added to SUMOylation reaction mixtures containing His₆-SUMO1, His₆-Ubc9, SAE1/His₆SAE2 (2.25 ng/µl), p45/p18 or p45 alone and ATP, which were then incubated at 37°C for 60 min. The reactions were then boiled in SDS sample buffer and separated by 11.25% and 12.5% SDS-PAGE, followed by immunoblotting with anti-p45 and p18 antibody, respectively.</p

CDK1/cyclin B enhances SUMOylation level of human TOP1 containing multiple SUMO conjugation site.

<p>(A) Full-length protein of human topoisomerase I was incubated with His₆-SUMO1, His₆-Ubc9, SAE1/His-SAE2 and ATP in the presence of 6, 12, 30 and 60 nM of CDK1/cyclin B. The reaction was resolved on a 6% SDS-PAGE and immunoblotted with anti-topoisomerase I antibody. (B) Equal amount of CDK1/cyclin B phosphorylated Ubc9 (P-E2) and Ubc9 (E2) (6 µM at the final concentration) was individually incubated with His₆-SUMO1, SAE1/His₆-SAE2, GST-hTOP1^(1–200) and ATP at 37°C for 30 min, 60 min and 120 min. The resulting SUMOylation reaction mixtures were run on a 8% SDS-PAGE, followed by immunoblotting with anti-topoisomerase I antibody. The amount of phosphorylated Ubc9 (P-E2) and Ubc9 (E2) for SUMOylation assay in Fig. 5B was determined by immunoblotting with anti-Ubc9 antibody. Lane 1 and 3 (right panel) were loaded with 1 µg Ubc9 and phosphorylated Ubc9, respectively. Lane 2 and lane 4 (right panel) were loaded with 2 µg of Ubc9 and phosphorylated Ubc9, respectively.</p

CDK1/cyclin B upregulates SUMOylation by increasing thioester bond formation between phosphorylated Ubc9 and SUMO1 but not between SAE1/SAE2 and SUMO1.

<p>(A) To examine the thioester bond formation between His₆-SUMO1 and SAE1/His₆-SAE2, purified His₆-SUMO1 and SAE1/His₆-SAE2 were incubated with various concentrations of CDK1/cyclin B (1.5 nM, 3 nM and 6 nM) in the presence or absence of ATP. Reaction mixtures were run on a 10% SDS-PAGE and immunoblotted with anti-SAE1/SAE2 antibody. (B) Ubc9 thioester conjugation assay was performed by incubating purified His₆-Ubc9, SAE1/His₆-SAE2 and His₆-SUMO1 with various concentrations of CDK1/cyclin B (1.2 nM, 3 nM and 6 nM) in the presence of ATP for 60 min at 37°C. Samples were then resolved on a 15% SDS-PAGE and probed with anti-Ubc9 antibody. (C) Ubc9 thioester conjugation assay in was performed similar as in (B), but at the end of the reaction, 200 mM of DTT was added to samples for overnight at 40°C. The reduced samples were run on a 15% SDS-PAGE, followed by immunoblotting with anti-Ubc9 antibody. Iso, isopeptide bond; Thiol, thioester bond.</p

CDK1/cyclin B enhances SUMOylation activity through wild type Ubc9 but not mutant Ubc9 S70A/S71A.

<p>Same amount of wild-type and mutant (S70A/S71A) of Ubc9 (at a concentration of 6 µM) was individually added to <i>in vitro</i> SUMOylation reaction mixtures containing His₆-SUMO1, SAE1/His₆-SAE2, GST-hTOP1 ^(110–125) AA and ATP in the presence of CDK1/cyclin B (6 nM and 12 nM) at 37°C for 60 min. The reactions were then boiled in SDS sample buffer and analyzed by 15% SDS-PAGE, followed by immunoblotting with anti-GST antibody.</p

CDK1/cyclin B-phosphorylated Ubc9 exhibits elevated SUMOylation activity.

<p>(A) Purified His₆-Ubc9 was incubated with GST-CDK1/cyclin B in the presence or absence of ATP for 30 min at 30°C. The GST-CDK1/cyclin B was then removed by passing through glutathione sepharose. The bound fraction of glutathione sepharose (contains phosphorylated Ubc9) and flow through fractions (FT, FT2, FT3) were analyzed by 12.5% SDS-PAGE, followed by immunoblotting with anti-GST and anti-Ubc9 antibody. (B) 3 µM, 6 µM and 12 µM of phosphorylated Ubc9 (flow through fraction 3) and non-phosphorylated Ubc9 (flow through fraction 3) were individually incubated with His₆-SUMO1, SAE1/His₆-SAE2, GST-hTOP1^(110–125) and ATP for 60 min at 37°C. The SUMOylation reaction mixtures were then analyzed by 15% SDS-PAGE, followed by immunoblotting with anti-GST antibody. The amount of phosphorylated and non-phosphorylated Ubc9 for SUMOylation assay were detected by immunoblotting with anti-Ubc9 antibody.</p