Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2

Brown, Nils; Lowe, E.D.; Petri, Ed; Skamnaki, V.T.; Antrobus, Robin; Johnson, L.N.

doi:10.4161/cc.6.11.4278

Cited by 83 publications

(86 citation statements)

References 42 publications

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“…CDK4/cyclin D3 and T160pCDK2/cyclin A or T160pCDK2 (100 ng unless otherwise stated) were mixed with 5 g of substrate (Histone H1, GST-RbCT, or GST-p107spacer), 0.1 mM ATP, 1 Ci [ 32 P]␥-ATP, 5 mM MgCl2, 50 mM Tris⅐HCl (pH 7.5) (final volume 10 L) at 20°C. After 7.5 min, the reactions were stopped by the addition of SDS and samples were analyzed by SDS/PAGE and autoradiography (32). p27 Kip1 was included as appropriate.…”

Section: Methodsmentioning

confidence: 99%

“…Human CDK1, CDK4, and cyclin D3 were all expressed in insect cells. CDK1 was expressed as a GST fusion and purified as described (32). CDK4 and cyclin D3 were cloned into pFastBac1 (Invitrogen) with, respectively, GST and PreScission protease recognition sequences and a FLAG tag at the amino terminus, and they were coexpressed in Sf-9 cells and purified by exploiting the CDK4 GST tag.…”

Section: Methodsmentioning

confidence: 99%

“…The activity of CDK1 or CDK2 toward recruited substrates has been shown to depend on the identity of the associated cyclin subunit, the local epitope around the site of phospho-transfer (respectively, either a canonical S/TPXR/K or noncanonical S/TPXX motif) (31,32), and the distance between the RXL⌽ and S/TPXK/R motifs (33). In particular, the RXL motif has been found to make a poor CDK substrate a better one, an effect that is enhanced if the substrate lacks a basic residue at Pϩ3 (refs.…”

Section: Cdk4/cyclin D3 Can Be Expressed In Insect Cells As An Activementioning

confidence: 99%

See 2 more Smart Citations

The structure of CDK4/cyclin D3 has implications for models of CDK activation

Takaki¹,

Echalier

Brown³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

109

123

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Cyclin-dependent kinase 4 (CDK4)/cyclin D complexes are expressed early in the G 1 phase of the cell cycle and stimulate the expression of genes required for G 1 progression by phosphorylation of the product of the retinoblastoma gene, pRb. To elaborate the molecular pathway of CDK4 activation and substrate selection we have determined the structure of nonphosphorylated CDK4/ cyclin D3. This structure of an authentic CDK/cyclin complex shows that cyclin binding may not be sufficient to drive the CDK active site toward an active conformation. Phosphorylated CDK4/cyclin D3 is active as a pRb kinase and is susceptible to inhibition by p27 Kip1 . Unlike CDK2/cyclin A, CDK4/cyclin D3 can be inactivated by treatment with -phosphatase, implying that phosphorylated T172 is accessible to a generic phosphatase while bound to a cyclin. Taken together, these results suggest that the structural mechanism of CDK4/cyclin D3 activation differs markedly from that of previously studied CDK/cyclin complexes.cell cycle ͉ kinase ͉ phosphorylation ͉ X-ray crystallography ͉ G1 phase T he transition of cells through the early G 1 stage of the cell cycle is coordinated by the activities of cyclin-dependent kinase 4 (CDK4) and CDK6 complexes that are formed after the mitogen-dependent expression of cyclin D. CDK4/cyclin D and CDK6/cyclin D stimulate the expression of genes required for G 1 progression by phosphorylation of the product of the retinoblastoma gene, pRb (reviewed in ref. 1). In wild-type cells it is considered that CDK2, which is found associated first with cyclin E and then with cyclin A, subsequently coordinates events as cells progress from G 1 through S phase (reviewed in ref.2). This model of sequential CDK activation controlling cell cycle progression has been elaborated by studies of CDK and cyclin knockout mice that showed that various mouse cell types derived from knockout animals are able to proliferate in the absence of several CDKs. This work further revealed that, although the CDK and cyclin families exhibit considerable functional redundancy, certain cell cycle CDKs have essential cell-type specific roles through their ability to phosphorylate specific substrates (3) or bind to specific cyclins (2).The principal CDK substrate during early G 1 is pRb. Sequential CDK-dependent pRb phosphorylation results in dissociation of pRb-transcription regulator complexes and concomitant enhancement of expression of genes whose products are required for G 1 progression (4, 5). pRb is initially phosphorylated on multiple sites by CDKs 4 and 6 and then additionally phosphorylated by CDK2 bound to cyclins E and A (6-8). Few authentic CDK4 substrates have been identified: CDK4 phosphorylates members of the pRb family (9) and the transcription factor Smad3 (10) but does not phosphorylate the canonical CDK substrate histone H1 (9,11,12) or the cyclin-dependent kinase inhibitor (CKI) p27 Kip1 (13).A model for CDK regulation by cyclin and CKI binding and by phosphorylation has been developed from a series of structures of CDK2/cycli...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cdk4/cyclin D3 Can Be Expressed In Insect Cells As An Activementioning

confidence: 99%

See 1 more Smart Citation

The structure of CDK4/cyclin D3 has implications for models of CDK activation

Takaki¹,

Echalier

Brown³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

109

123

View full text Add to dashboard Cite

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“…Surprisingly, knockdown of Gpr19 affected progression through the cell-cycle at a later stage, namely in G 2 -M. This conclusion is based on 2 independent lines of evidence, namely the determination of DNA content by flow cytometry and the quantification of the G 2 -M marker cyclin B1 and the mitosis marker phosphorylated histone H3 by immunocytochemistry. Cyclin B1 exerts its function in conjunction with cyclin-dependent kinase 1 during late G 2 -phase and early mitosis (44). Histone H3 phosphorylation at Ser10 correlates with chromosome condensation during mitosis with phosphorylation levels starting to increase in late G 2 -phase (45).…”

Section: Discussionmentioning

confidence: 99%

Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry into S-Phase and Supports G2–M Cell-Cycle Progression

Kastner

Voss

Keuerleber

et al. 2012

Molecular Cancer Research

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It has long been known that G protein-coupled receptors (GPCR) are subject to illegitimate expression in tumor cells. Presumably, hijacking the normal physiologic functions of GPCRs contributes to all biologic capabilities acquired during tumorigenesis. Here, we searched for GPCRs that were expressed in lung cancer: the mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found frequently overexpressed in tissue samples obtained from patients with small cell lung cancer. Several observations indicate that overexpression of Gpr19 confers a specific advantage to lung cancer cells by accelerating transition through the cellcycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines. (ii) Cell-cycle progression through G 2 -M-phase was impaired in cells transfected with siRNAs directed against Gpr19 and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. (iii) The expression levels of Gpr19 mRNA varied along the cell-cycle with a peak observed in S-phase. (iv) The putative control of Gpr19 expression by E2F transcription factors was verified by chromatin immunoprecipitation: antibodies directed against E2F-1 to -4 allowed for the recovery of the Gpr19 promoter. (v) Removal of E2F binding sites in the Gpr19 promoter diminished the expression of a luciferase reporter.

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“…[39][40][41][42] Very recently, two studies have reported the structures of cyclin B. [43][44] To begin with, we examine the crystal structure of cyclin A2 41 to establish the structural framework for peptide binding. This reveals an overall fold characterized by two a-helical domains (Fig.…”

Section: Introductionmentioning

confidence: 99%

Substrate Specificity of Cyclins Determined by Electrostatics

Lee

Chua²,

Krishnan³

et al. 2007

Cell Cycle

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ACKnowLeDGementSBII and IMCB are A-STAR institutes funded by BMRC. We thank Michael Schaefer for providing us with unpublished parameters for version 3 of the analytical continuum solvent method. AbStrACtCyclin dependent kinases (CDK) associate with cyclins to regulate cell cycle progression and gene transcription by phosphorylating key proteins. The different cyclin-CDK complexes display differences in substrate specificities with substrates binding across a shallow, hydrophobic and substrate-binding pocket known as the cyclin groove. However the mechanism underlying this differential substrate recognition remains largely unknown and cannot be explained merely on the basis of sequence variability. A subset of cyclins, cyclins A2, E1 and B1 despite being structurally and functionally similar, show marked differences in their interactions with recruitment peptides derived from their substrate or inhibitor proteins p27, p21, p57, E2F1, p53, pRb and p107. While these peptides (characterized by a cyclin binding motif of four residues ZRXL where Z and X are cationic residues) inhibit the activity of cyclins A2 and E1, no such inhibition is observed for cyclin B1.Electrostatic potentials of cyclins A2, E1 and B1 show that anionic regions of cyclins A2 and E1 enable them to bind peptides while cationic regions at homologous locations in cyclin B1 abrogate binding. These arise from charged residues that are conserved. Mutations that switch these characters are suggested. Computed energetics of binding confirms this. Deregulation of the enzymatic activity of this class of enzymes is a ubiquitous feature of human neoplasia, but attempts to exploit this therapeutically have been confounded by a lack of understanding of the precise specificity of the different cyclin complexes. Here we begin to clarify this issue by explaining the mechanism by which cyclin B1 escapes regulation by the p21 family of CDKIs.

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Cyclin B and Cyclin A Confer Different Substrate Recognition Properties on CDK2

Cited by 83 publications

References 42 publications

The structure of CDK4/cyclin D3 has implications for models of CDK activation

The structure of CDK4/cyclin D3 has implications for models of CDK activation

Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry into S-Phase and Supports G2–M Cell-Cycle Progression

Substrate Specificity of Cyclins Determined by Electrostatics

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