Dual Regulation of the Anaphase Promoting Complex in Human Cells by Cyclin A-Cdk2 and Cyclin A-Cdk1 Complexes

Mitra, Jayashree; Enders, Greg H.; Azizkhan‐Clifford, Jane; Lengel, Kathleen L.

doi:10.4161/cc.5.6.2604

Cited by 25 publications

(28 citation statements)

References 22 publications

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“…Since SFETP SLBP was expressed at normal levels during S phase and then degraded, we conclude that phosphorylation of Thr61 by cyclin A/Cdk1 is essential for proper timing of the degradation of SLBP. In support of this, it has been shown that cyclin A/Cdk1 is activated at the end of S phase (21,22). Thus, the mutagenesis data taken together with our biochemical results implicate cyclin A/Cdk1 as the kinase that phosphorylates Thr61 of SLBP, and this phosphorylation triggers degradation of SLBP at the end of S phase.…”

Section: Resultssupporting

confidence: 58%

“…Although there is high cyclin A/Cdk2 activity throughout S phase, both the in vitro Thr61 kinase activity in cell lysates and the SLBP degradation in vivo do not occur until the S/G 2 border. Thus, the Thr61 phosphorylation profile we detected correlates well with cyclin A/Cdk1 activity which increases toward late S phase, with a gradual increase toward the point where it peaks at G 2 (21,22).…”

Section: Discussionmentioning

confidence: 75%

“…There is an increase in cyclin A/Cdk1 activity at the end of S phase, and it increases prior to the activation of cyclin B/Cdk1 (21,22). This pattern of activity is distinct from that of cyclin A/Cdk2, which is active throughout S phase and increases only slightly during G 2 (22,24,26).…”

Section: Discussionmentioning

confidence: 85%

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Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

Köseoğlu¹,

Graves²,

Marzluff

2008

Molecular and Cellular Biology

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Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G 2 .Progression through the cell cycle is driven by a class of protein kinases, the cyclin-dependent kinases (cdk's), which are sequentially activated as cells progress from one cell cycle stage to the next. In particular, progress into S phase requires the activation of cyclin E/Cdk2, ultimately resulting in the initiation of DNA replication, and progression through mitosis requires the activation of cyclin B/Cdk1 (cdc2), resulting in nuclear envelope breakdown and chromosome condensation (27). Cyclin A/Cdk2 activity is required for continued progression through S phase (36). The targets of the cdk's are activated or inactivated by phosphorylation. A second critical regulatory mechanism for cell cycle progression is targeted proteolysis of key protein regulators (13). These include the cyclin subunits of the cdk's, critical proteins in initiation of DNA replication, and proteins responsible for maintaining chromosome pairing (6).While much is known about the events that must occur for cells to transit from G 1 to S phase and for cells to enter and exit mitosis, much less is known about the molecular events at the end of S phase, as cells progress from S to G 2 phase. S phase is characterized by replication of the chromosomes, and at the same time DNA is replicated, histone proteins must be synthesized to provide histones to assemble the newly replicated chromatin. Histone mRNAs are cell cycle regulated, and their expression is restricted to S phase. The metazoan replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Instead, they end in a conserved stem-loop sequence (18). Since the replication-dependent histone genes lack introns, the only processing reaction required for histone mRNA biosynthesis is cleavage of the nascent transcript to ...

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Section: Resultssupporting

confidence: 58%

Section: Discussionmentioning

confidence: 75%

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Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

Köseoğlu¹,

Graves²,

Marzluff

2008

Molecular and Cellular Biology

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“…We first asked whether endogenous Cdk2 and Wee1 are physically associated. Cdk2 is the major Cdk partner of cyclin A in human cells and does not bind appreciably to cyclin B in the presence of Cdk1 (21,35,37,38). Cdk2 was readily detected in somatic Wee1 immunoprecipitates (IPs) derived from asynchronous cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Because Cdk2 binds most cyclin A present in U2-OS cells during late S and G 2 phases (37), most Wee1 bound to cyclin A is likely in complex with Cdk2. Some complexes may also contain Cdk1, which associates with a low but increasing fraction of cyclin A as U2-OS cells approach mitosis (38). (18).…”

Section: Resultsmentioning

confidence: 99%

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Andrake

Dunbrack

et al. 2010

Molecular and Cellular Biology

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Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G 2 /M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G 2 /M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.

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β‐catenin inhibitor ICG‐001 suppress cell cycle progression and induce autophagy in endometrial cancer cells

Hsin,

Wu,

Tang

et al. 2023

Journal Cellular Physiology

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The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of β‐catenin are commonly detected in endometrioid endometrial cancer. ICG‐001 is a β‐catenin inhibitor via blocking the complex formation of β‐catenin and cAMP response element‐binding protein (CREB)‐binding protein (CBP). This study aims to investigate the effect of ICG‐001 on endometrial cancer inhibition. First, endometrial carcinoma patient‐derived xenograft (PDX)‐derived organoids and primary cells were used to verify the inhibiting ability of ICG‐001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG‐001. Using MTT assay and tumor spheroid formation assay, ICG‐001 significantly reduced the cell viability of HEC‐59 and HEC‐1A cells. ICG‐001 enhanced cisplatin‐mediated cytotoxicity. ICG‐001 decreased cancer stem cell sphere formation. ICG‐001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG‐001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG‐001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG‐001‐mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG‐001 against endometrial cancer.

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Dual Regulation of the Anaphase Promoting Complex in Human Cells by Cyclin A-Cdk2 and Cyclin A-Cdk1 Complexes

Abstract: We are grateful to Jiri Lukas (Danish Cancer Society, Denmark) for providing us with the wild type Cdh1-expressing U2-OS cell line and the Cdh1 mutant (RVL-AAA) plasmid. We thank Andrew Ippolito for editorial assistance.

Cited by 25 publications

References 22 publications

Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

β‐catenin inhibitor ICG‐001 suppress cell cycle progression and induce autophagy in endometrial cancer cells

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