Activation of human cyclin-dependent kinases in vitro.

Desai, Dev M.; Gu, Yun; Morgan, David

doi:10.1091/mbc.3.5.571

Cited by 244 publications

(191 citation statements)

References 43 publications

Supporting

Mentioning

180

Contrasting

Order By: Relevance

“…Although the titer of cyclin varied slightly between batches, in all cases maximal inhibition was observed using <0.2 ,tM cyclin A. This concentration is lower than that of cyclin B used in a similar study (Thomas et al, 1992) and compares both with the concentration of cdc2 found in the cell (see Desai et al, 1992) and with levels of cyclin A required to induce meiosis (Roy et al, 1991) or activate cdc2 kinase (Clarke et al, 1992) in Xenopus. We also compared cyclin Aactivated cytosols with cytosols prepared from mitotic cells that contained elevated levels of cyclin B-cdc2 kinase.…”

Section: Inhibition Of Fusion Is Reversed By Addition Of Untreated Cymentioning

confidence: 62%

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Woodman

Adamczewski

Hunt

et al. 1993

MBoC

View full text Add to dashboard Cite

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

show abstract

Section: Inhibition Of Fusion Is Reversed By Addition Of Untreated Cymentioning

confidence: 62%

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Woodman

Adamczewski

Hunt

et al. 1993

MBoC

View full text Add to dashboard Cite

show abstract

“…Activation of CDK6 by KSHV-cyclin is unconventional and very strong. So far all cell cycle cdk/cyclin complexes examined have required phosphorylation of the activating threonine within the T-loop by CAK for full activity (Gould et al, 1991;Desai et al, 1992;Solomon et al, 1992; ConnellCrowley et al, 1993;Fisher and Morgan, 1994;Kato et al, 1994;Matsuoka et al, 1994;Aprelikova et al, 1995;Kaldis et al, 1996. The only exception is CDK7 and its orthologs, where binding of the assembly factor MAT1 to the CDK7/cyclin H complex can substitute for activating phosphorylation Kimmelman et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…Phosphorylation of an activating threonine (Thr-160 in CDK2 and Thr-177 in CDK6) by the cdk-activating kinase (CAK; reviewed by Kaldis, 1999) is accompanied by structural changes in the T-loop (also called the activation segment), allowing the phosphate group to interact with several other residues and thereby acting as an organizing center in the catalytic cleft (Russo et al, 1996b). Mutation of the acti- vating threonine to an unphosphorylatable amino acid prevents activation of vertebrate cdks (Desai et al, 1992;Solomon et al, 1992;Connell-Crowley et al, 1993;Kato et al, 1994;Matsuoka et al, 1994) and equivalent mutants are unable to support growth in yeast (Gould et al, 1991;Cismowski et al, 1995). Two very different CAKs have been identified.…”

mentioning

confidence: 99%

CAK-independent Activation of CDK6 by a Viral Cyclin

Kaldis

Ojala

Tong

et al. 2001

MBoC

View full text Add to dashboard Cite

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16 INK4a . Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16 INK4a sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6 T177A together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK. INTRODUCTIONThe sequential activation of cyclin-dependent kinases (cdks) promotes cell cycle transitions. CDK4 and CDK6 bind to D-type cyclins and are active in G1, CDK2/cyclin E complexes function in late G1, CDK2/cyclin A complexes function in S phase, and CDC2/cyclin B and A complexes function in G2/M. The activities of cdks are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations (Pines, 1995;Sherr and Roberts, 1995;King et al., 1996;Solomon and Kaldis, 1998). Viral infection frequently targets downstream targets of cdks, resulting in inappropriate cell cycle progression.Cyclin binding activates cdks by inducing conformational changes in the structure of cdks (Jeffrey et al., 1995;Pavletich, 1999). Cyclins are unstable proteins that are synthesized and degraded periodically during the cell cycle. Transcriptional control (Koch and Nasmyth, 1994) and ubiquitin-mediated degradation (King et al., 1996) ensure the proper and irreversible timing of cell cycle regulatory events. Cyclins have also been shown to affect the substrate specificity of cdks (Peeper et al., 1993;Kelly et al., 1998;Schulman et al., 1998;Cross et al., 1999).Maximal activation of cdks requires phosphorylation of certain residues as well as dephosphorylation of others. The dual-specificity phosphatase CDC25 removes phosphates from inhibitory phosphorylation sites (Thr-14 and Tyr-15 in human CDK2) that have been phosphorylated by the WEE1/MYT1 protein kinases . Phosphorylation of an activating threonine (Thr-160 in CDK2 and Thr-177 in CDK6) by the cdk-activating kinase (CAK; reviewed by Kaldis, 199...

show abstract

“…Soluble glutathione S-transferase (GST)-cyclin A/Cdk2, GST-cyclin E/Cdk2, GST-cyclin E/His6-Cdk3, and cyclin D/GST-Cdk4 complexes were produced in insect cells (High5, Invitrogen, San Diego, CA) and purified on glutathione-Sepharose (GSH-Sepharose, Pharmacia, Uppsala, Sweden) using established procedures (Desai et al, 1992;Matsushime et al, 1992;Harper et al, 1995). The sources of the baculovirus are as described (Harper et al, 1995) N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), pH 7.6, 150 mM NaCl, 1 mM EDTA, 2.5 mM ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 % Tween 20, 1 mM dithiothreitol, 5 mM NaF, 30 mM p-nitrophenyl phosphate, 25 mM (3-glycerol phosphate, 1 ,ug/ml leupeptin, 1 ,ug/ml antipain, and 1 mM PMSF] for Cdk4 complexes.…”

Section: Protein Purificationmentioning

confidence: 99%

Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.

Connell-Crowley

Harper

Goodrich

1997

MBoC

386

352

View full text Add to dashboard Cite

The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin Dl /Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A-or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating "hyperphosphorylated form." The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous. INTRODUCTIONThe retinoblastoma protein (pRb) functions to constrain cell proliferation and exerts its effects during the

show abstract

Activation of human cyclin-dependent kinases in vitro.

Cited by 244 publications

References 43 publications

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

CAK-independent Activation of CDK6 by a Viral Cyclin

Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.

Contact Info

Product

Resources

About