The N-terminal Helix of Xenopus Cyclins A and B Contributes to Binding Specificity of the Cyclin-CDK Complex

Goda, Tadahiro; Funakoshi, Mitsuaki; Suhara, Hiroto; Nishimoto, Tetsuya; Kobayashi, Hideki

doi:10.1074/jbc.m011101200

Cited by 18 publications

(20 citation statements)

References 38 publications

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“…In the case of the Cdk2 K278A mutant, no direct contacts with the T-loop have been described, and this mutation may directly affect the conformational change induced upon interaction with cyclin A. Lys 278 was found to form a side-chain hydrogen bond at the Cdk2/cyclin A and Cdk2/cyclin M interface with residues Asp 181 and Tyr 178 in the cyclins (4,8). These results are in agreement with the finding that the mutation of Ser 277Asp in Xenopus Cdc2 compensates for a Thr 161Ala mutation and induce oocyte maturation (16), and that the mutation Ala 280Asn in Xenopus Cdk2 abolishes the binding of cyclin A (17), suggesting that the conformation of the C-lobe is important to maintain the structure of the activation loop in Xenopus Cdc2 and Cdk2. The recent structure of the ␥-Herpesvirus cyclin M in complex with Cdk2 reveals that the stability of the complex is increased compared with that of Cdk2/cyclin A because of extended contacts between the C-lobe of Cdk2 and the N-terminal helix of the cyclin.…”

Section: The Pstaire Motif Of the Cdk Is Essential For The Initial Assupporting

confidence: 82%

Kinetic Mechanism of Activation of the Cdk2/Cyclin A Complex

Morris¹,

Gondeau²,

Tainer³

et al. 2002

Journal of Biological Chemistry

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Eukaryotic cell cycle progression is controlled by the ordered action of cyclin-dependent kinases, activation of which occurs through the binding of the cyclin to the Cdk followed by phosphorylation of a conserved threonine in the T-loop of the Cdk by Cdk-activating kinase (CAK). Despite our understanding of the structural changes, which occur upon Cdk/cyclin formation and activation, little is known about the dynamics of the molecular events involved. We have characterized the mechanism of Cdk2/cyclin A complex formation and activation at the molecular and dynamic level by rapid kinetics and demonstrate here that it is a two-step process. The first step involves the rapid association between the PSTAIRE helix of Cdk2 and helices 3 and 5 of the cyclin to yield an intermediate complex in which the threonine in the T-loop is not accessible for phosphorylation. Additional contacts between the C-lobe of the Cdk and the N-terminal helix of the cyclin then induce the isomerization of the Cdk into a fully mature form by promoting the exposure of the T-loop for phosphorylation by CAK and the formation of the substrate binding site. This conformational change is selective for the cyclin partner.Eukaryotic cell cycle progression is governed by members of the cyclin-dependent kinase family (Cdks), 1 heterodimeric complexes consisting of a catalytic Ser/Thr protein kinase subunit, Cdk, and of a regulatory cyclin subunit. The activation of monomeric Cdk subunits involves the binding of a cyclin partner, which confers basal kinase activity to the Cdk/cyclin complex and enables subsequent phosphorylation of the Cdk on a conserved threonine in the activation loop (Thr 160 in Cdk2), thereby finally converting the complex into a fully active form (1, 2). The determination of the structure of unphosphorylated and phosphorylated Cdk2/cyclin A complexes has revealed that cyclin binding induces conformational changes within Cdk2 that are critical for its activation (3-6). The most significant feature is the reconfiguration of the ATP binding site into a conformation that favors its nucleophilic attack by the substrate and brings Glu 51 together with Lys 33 and Asp 145 for catalysis. In addition, cyclin binding induces a positional switch of the T-loop by 20 Å, which opens the catalytic cleft, affects the orientation of the putative substrate binding site of Cdk2, and leads to appropriate exposure of Thr 160 . Subsequent phosphorylation of Thr 160 by Cdk7/cyclin H (CAK) induces further conformational changes in the T-loop and in the Cterminal lobe of Cdk2 and stabilizes the substrate binding site (5, 6). More recently, the determination of the crystal structures of Cdk6/cyclin K (7), Cdk2/cyclin M (8), and Cdk2/Kap (9) suggest that interactions involving the C-lobe of Cdks play an important role in activation and regulation of the latter.Although the structural changes involved in Cdk/cyclin formation and activation have been identified, the mechanism and dynamics of the molecular events underlying this process are still poorly unders...

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Section: The Pstaire Motif Of the Cdk Is Essential For The Initial Assupporting

confidence: 82%

Kinetic Mechanism of Activation of the Cdk2/Cyclin A Complex

Morris¹,

Gondeau²,

Tainer³

et al. 2002

Journal of Biological Chemistry

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“…1A). The CBF repeat is required for cyclins to bind to Cdks (Jeffrey et al, 2000) and the L×C×E motif contributes the binding site for the pRb substrate , whereas the N-terminal helix confers a second step of cyclin and Cdk interaction and exposure of the T-loop of the Cdk for phosphorylation (Card et al, 2000;Goda et al, 2001;Morris et al, 2002). Therefore, the truncated D2C4 complex would not be able to bind to and phosphorylate pRb or initiate DNA synthesis.…”

Section: Cyclin D2 Interacts With Cdk4 In Vivomentioning

confidence: 99%

Migratory Localization of Cyclin D2-Cdk4 Complex Suggests a Spatial Regulation of the G1-S Transition

Wang

Xie

Zhang

et al. 2008

Cell Struct. Funct.

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ABSTRACT. The association of the cyclin D-Cdk (DC) complex with retinoblastoma protein (pRb) is required for the G1-S transition of the cell cycle. Cyclin synthesis, nuclear localization and degradation are control mechanisms for the transition, but regulation of the DC complex nuclear import also contributes to the transition. Analysis of the timing of the G1-S transition in mammalian cell lines revealed acceleration with overexpression of cyclin D2 and Cdk4. Immunolocalization assays revealed that cyclin D2 and Cdk4 formed a complex in the cytoplasm and approached the nucleus. They accumulated on the cytosolic surfaces of the nuclear pores and then were arrested at the nuclear membrane before the nucleus reached a critical size. Finally, the complex was released into the nucleus and colocalized with pRb there, which led to pRb phosphorylation and DNA synthesis. The translocalization depended on the G1-S transition. In contrast, a truncated cyclin D2 that was not able to fully associate with Cdk4 lost the ability for release into the nucleus. This pattern of translocalization suggests a spatial separation of the cyclin D-Cdk complex from pRb and DNA in the nucleus to regulate the G1-S transition.

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“…We tested three truncated forms of cyclin B in this way (Fig. 1C): MBP-CBNT contains the N-terminal domain of cyclin B that includes the destruction box (D-box) but excludes the two Cdc2-binding cyclin boxes; MBP-CBCT1 contains the Cterminal domain of cyclin B and includes the two cyclin boxes and the 'helix' domain that is thought to promote the specific interaction with cell division protein kinases (CDKs) (Goda et al, 2001); MBP-CBCT2 is a truncated version of MBP-CBC1 that is missing the helix domain. A western-blot analysis of this pull down experiment (Fig.…”

Section: Cyclin B Interacts With Hsp90mentioning

confidence: 99%

Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle inDrosophilaand humans

Basto

Gergely

Draviam

et al. 2007

Journal of Cell Science

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During mitosis, cyclin B is extremely dynamic and although it is concentrated at the centrosomes and spindle microtubules (MTs) in organisms ranging from yeast to humans, the mechanisms that determine its localisation are poorly understood. To understand how cyclin B is targeted to different locations in the cell we have isolated proteins that interact with cyclin B in Drosophila embryo extracts. Here we show that cyclin B interacts with the molecular chaperone Hsp90 and with the MT-associated protein (MAP) Mini spindles (Msps; the Drosophila orthologue of XMAP215/ch-TOG). Both Hsp90 and Msps are concentrated at centrosomes and spindles, and we show that Hsp90, but not Msps, is required for the efficient localisation of cyclin B to these structures. We find that, unlike what happens with other cell cycle proteins, Hsp90 is not required to stabilise cyclin B or Msps during mitosis. Thus, we propose that Hsp90 plays a novel role in regulating the localisation of cyclin B and Msps during mitosis.

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The N-terminal Helix of Xenopus Cyclins A and B Contributes to Binding Specificity of the Cyclin-CDK Complex

Cited by 18 publications

References 38 publications

Kinetic Mechanism of Activation of the Cdk2/Cyclin A Complex

Kinetic Mechanism of Activation of the Cdk2/Cyclin A Complex

Migratory Localization of Cyclin D2-Cdk4 Complex Suggests a Spatial Regulation of the G1-S Transition

Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle inDrosophilaand humans

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