Inhibition of CDK1 activity by sumoylation

Abstract

Sumoylation (a covalent modification by Small Ubiquitin-like Modifiers or SUMO proteins) has been implicated in the regulation of various cellular events including cell cycle progression. We have recently identified CDK1, a master regulator of mitosis and meiosis, as a SUMO target both in vivo and in vitro, supporting growing evidence concerning a close cross talk between sumoylation and phosphorylation during cell cycle progression. However, any data regarding the effect of sumoylation upon CDK1 activity have been missing. In this study, we performed a series of in vitro experiments to inhibit sumoylation by three different means (ginkgolic acid, physiological levels of oxidative stress, and using an siRNA approach) and assessed the changes in CDK1 activity using specific antibodies and a kinase assay. We have also tested for an interaction between SUMO and active and/or inactive CDK1 isoforms in addition to having assessed the status of CDK1-interacting sumoylated proteins upon inhibition of sumoylation. Our data suggest that inhibition of sumoylation increases the activity of CDK1 probably through changes in sumoylated status and/or the ability of specific proteins to bind CDK1 and inhibit its activity.

Keywords: CDK1; Ginkgolic acid; H(2)O(2); Phosphorylation; Sumoylation; siRNA.

Figure 1. Inhibition of sumoylation increases CDK1 activity

HEK and GC1 cells were incubated with A) sumoylation inhibitor ginkgolic acid (200 μM GA, 6 hours), B) H₂O₂, 2 mM; 1 hour, and C) UBC9 (SUMO-conjugating enzyme) siRNA as described in the materials and methods section, followed by a western blot analysis using an anti-SUMO and -an inhibitory (Tyr 15) phosphorylated or total CDK1 isoform antibodies. Inhibition of sumoylation (a decrease in High Molecular Weight (HMW) SUMO conjugates) was confirmed using anti-SUMO1 (shown) and SUMO 2/3 (not shown) antibodies that produced similar patterns on the western blot. Equal loading was confirmed using anti-actin antibody. Three different representative experiments are shown for each treatment. HEK data are shown in A) and B), and GC1 data are shown in C). Similar results were obtained in HEK and GC1 cell lines. To calculate the difference between the samples (n=5), a student’s paired t-test was used. P values <0.05 were considered statistically significant.

Figure 2. Kinase assay

An immunoprecipitation with anti-CDK1 antibody was followed by a kinase assay using CDK substrate with control and UBC9 down-regulated samples. Successful specific enrichment for CDK1 was confirmed using a western blot with anti-CDK1 antibody (A). The kinase assay measures the amount of free ATP left after the reaction (Fig. 2B, Y-axis) and is thus inversely related to kinase activity (B). To calculate the difference between the samples (n=3), a student’s paired t-test was used. P values <0.05 were considered statistically significant.

Figure 3

(A): Inactive CDK1 isoform is enriched in SUMO pull-down fraction. An immunoprecipitation of HEK cell lysates with anti-SUMO antibodies or IgG control was followed by a western blot with either anti-an inhibitory (Tyr 15) phosphorylated or total CDK1 isoform antibodies on the same membrane. (B): Inhibition of sumoylation decreases sumoylated status of CDK1-interacting proteins. An immunoprecipitation with an anti-CDK1 antibody or IgG control using the control HEK lysate and the lysate produced after an inhibition of sumoylation with 2 mM of H₂O₂ was followed by a western blot with anti-SUMO antibody. The arrow indicates sumoylated CDK1; and the bracket indicates high molecular weight (HMW) proteins interacting with CDK1 and detected in the SUMO pool-down fraction.