The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition

Abstract

Clb2 mitotic cyclin inhibits cell cycle progression by preventing mitotic exit and DNA synthesis. To allow cell cycle progression, Clb2 proteolysis is triggered by Cdc20 during the metaphase-to-anaphase (M-A) transition and by Hct1 during mitotic exit and G1 [1-6]. A cis element called the destruction box is required for this proteolysis [7-11]. Recently, an additional cis element called the "KEN box" was also shown to be required for proteolysis of human CDC20 and Securin [3,12]. Using a novel color assay, we show that a Clb2 KEN box is required to target a fusion protein containing the first 124 amino acids of Clb2 for proteolysis. We further show that full-length Clb2 bearing mutations in the KEN box is degraded efficiently during the M-A transition, but poorly during G1. If the destruction box of Clb2 is mutated in combination with mutation of the KEN box, then this form of Clb2 is more stable than Clb2 bearing either mutation by itself during both M-A and G1. Our results show that the KEN box and the destruction box act together during both M-A and G1 to regulate Clb2 proteolysis.

Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis

“…Clb2 remained stable in the G 1 phase in the cdh1 dbr and cdh1 kbr strains containing mutations in the D-box receptor and KENbox receptor, respectively, but was degraded in the cdh1 amr strain containing mutations in the ABBA motif receptor ( Fig. 2A), consistent with the presence of functional D-and KENboxes in Clb2 that direct its APC/C-mediated degradation (46,47). Stabilization of endogenous Clb2 was not due to differences in expression level of the mutant proteins (Fig.…”

A Novel RING Finger Protein, Human Enhancer of Invasion 10, Alters Mitotic Progression through Regulation of Cyclin B Levels

“…The Clb2p-⌬box mutant (20), lacking both a cyclin destruction box and a KEN box, shows a slow-growth phenotype due to the cell's very limited ability to degrade this mutant form of Clb2p. To further probe genetic interactions of HEI10 with Clb2p, we examined the effect of HEI10 or HEI10 derivatives on the growth of yeast coexpressing the Clb2p-⌬box mutant (Fig.…”

“…Certainly, many researchers have investigated the regulation of cyclin B proteolysis by the anaphase-promoting complex in detail without having previously identifyied HEI10. However, recent studies demonstrating combined action of multiple different destruction motifs for APC/C-mediated degradation in vivo have reopened the question of proteolytic targeting controls (18,20,33,59). It is becoming clear that important cell cycle regulatory proteins are degraded through the use of different targeting proteins at different points throughout the cell cycle.…”

Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos

“…The degradation of cyclin B1 promotes mitotic exit and contributes to the activation of separase in higher eukaryotes as well, where separase is inhibited by Cdk1/cyclin B1 (5,10,25). The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates (7,9,13,20,22). Destruction boxes or KEN boxes are motifs frequently found in APC/C's substrates, but other motifs are also possible for the recognition by APC/C-Cdc20 or APC/C-Cdh1 (7).…”