The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism

Clare, Paula M.; Poorman, Roger A.; Kelley, Laura C.; Watenpaugh, Keith David; Bannow, Carol A.; Leach, Karen L.

doi:10.1074/jbc.m102034200

Cited by 48 publications

(41 citation statements)

References 63 publications

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“…Consistent with these data it has been shown that active CDK4/CycD1 complex cannot be readily reconstituted by mixing recombinant CDK4 and cyclin D (2). Unlike CDK2/cyclin A (34), CDK4 does not appear to spontaneously activate when bound to cyclin D and exhibits ordered addition kinetics (35,36). Phosphorylation studies using peptidic or Rb-protein substrates reveal a low specific activity of CDK4/cyclin D1 versus peptides and a modulation of the enzymatic K m for ATP (36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of human CDK4 in complex with a D-type cyclin

Day¹,

Cleasby²,

Tickle³

et al. 2009

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

207

185

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The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G1 phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 Å. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation. CDK4 and CDK6 associate with the D-type cyclins (D1, D2, D3) and phosphorylate and inactivate the retinoblastoma (Rb) protein family members (p107, p130, pRb). Phosphorylation of pRb by CDK4/6 then leads to the derepression and activation of E2F target genes, including the E-type cyclins, which facilitate progression through the G 1 phase of the cell cycle.Deregulation of the CDK4/cyclin D pathway has been identified in many cancers (refs. 4 and 5 and references therein and ref. 6). Notably, most genetic alterations target specifically CDK4 or cyclin D1, whereas alterations in other CDKs and cyclins are far less common. The CDK4 gene is amplified in a high percentage of liposarcomas (7), and breast cancers frequently exhibit high cyclin D1 levels, either through genetic amplification of the gene or overexpression (8). Translocation of cyclin D1 to the IgH promoter is a hallmark aberration in mantle cell lymphoma (9). Cyclin D1 translocations can also be detected in many cases of multiple myelomas (10). A mutation of CDK4 (Arg-24-Cys) that renders it refractory to inhibition by the tumor suppressor protein p16INK4a has also been identified, and, similarly, deletion or mutation of the p16INK4a gene results in defective CDK4 inhibition and dysregulated CDK4 activity (11). Finally, genetic inactivation of p16INK4 is among the most frequent tumor suppressor mutations found in human cancers. Taken together, these data indicate that an unchecked or hyperactivated CDK4/cyclin D1 pathway may be responsible for enhanced cellular proliferation in cancers and imply that CDK4 is a promising target for the development of anticancer therapies (reviewed in ref. 12).The molecular basis of CDK activation has been the focus of many studies using cellular, biochemical, and structural approaches (reviewed in ref.3). Maximal CDK activation requires both binding of a cognate cyclin and phosphorylation of residues within the CDK T-loop, and X-ray crystallographic studies of various CDKs and CDK/cyclin complexes have identified the conformational movements associated with ...

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Section: Discussionmentioning

confidence: 99%

Crystal structure of human CDK4 in complex with a D-type cyclin

Day¹,

Cleasby²,

Tickle³

et al. 2009

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

207

185

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“…1, corresponding to unordered (random) substrate binding (10). [There is evidence that this is the mechanism driving at least some cyclin-dependent kinase-catalyzed phosphorylation reactions at the heart of the cell cycle (11). ]…”

Section: A Motivational Examplementioning

confidence: 99%

Understanding bistability in complex enzyme-driven reaction networks

Crăciun

Tang²,

Feinberg

2006

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

267

249

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Much attention has been paid recently to bistability and switch-like behavior that might be resident in important biochemical reaction networks. There is, in fact, a great deal of subtlety in the relationship between the structure of a reaction network and its capacity to engender bistability. In common physicochemical settings, large classes of extremely complex networks, taken with mass action kinetics, cannot give rise to bistability no matter what values the rate constants take. On the other hand, bistable behavior can be induced in those same settings by certain very simple and classical mass action mechanisms for enzyme catalysis of a single overall reaction. We present a theorem that distinguishes between those mass action networks that might support bistable behavior and those that cannot. Moreover, we indicate how switch-like behavior results from a well-studied mechanism for the action of human dihydrofolate reductase, an important anti-cancer target.bistability ͉ dihydrofolate reductase

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“…So far, numerous specific CDK inhibitors have been identified on the basis of their ability to inhibit CDK1, CDK2 or CDK4: the purines olomoucine (Vesely et al, 1994), roscovitine de Azevedo et al, 1997); purvalanols (Gray et al, 1998;Chang et al, 1999;Villerbu et al, 2002), CVT-313 (Brooks et al, 1997), C2-alkylynated purines (Legraverend et al, 2000), H717 (Dreyer et al, 2001) and NU2058 (Arris et al, 2000), piperidine-substituted purines (Shum et al, 2001), toyocamycin (Park et al, 1996), flavopiridol (Losiewicz et al, 1994), indirubins (Hoessel et al, 1999;Leclerc et al, 2001), paullones Zaharevitz et al, 1999;Leost et al, 2000), g-butyrolactone (Kitagawa et al, 1993), hymenialdisine , indenopyrazoles (Nugiel et al, 2001), the pyrimidines NY6027 (Arris et al, 2000) and CGP60474 (Zimmermann, 1995), pyridopyrimidines (Barvian et al, 2000), the aminopyrimidine PNU 112455A (Clare et al, 2001), oxindoles (Kent et al, 1999;Davis et al, 2001), PD0183812 (Fry et al, 2001), cinnamaldehydes (Jeong et al, 2000), quinazolines (Shewchuk et al, 2000;Sielecki et al, 2001), fasclaplysin (Soni et al, 2000(Soni et al, , 2001, SU9516 (Lane et al, 2001) and benzocarbazoles (Carini et al, 2001). Despite their chemical diversity, these inhibitors all act by competing with ATP at the ATP-binding site of the kinase.…”

Section: Introductionmentioning

confidence: 99%

p42/p44 MAPKs are intracellular targets of the CDK inhibitor purvalanol

et al. 2002

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Chemical inhibitors of cyclin-dependent kinases (CDKs) have a great therapeutic potential against various proliferative and neurodegenerative disorders. Intensive screening of a combinatorial chemistry library of 2,6,9-trisubstituted purines has led to the identification of purvalanol, one of the most potent and selective CDK inhibitors to date. In preliminary studies, this compound demonstrates definite anti-mitotic properties, consistent with its nanomolar range efficiency towards purified CDK1 and CDK2. However, the actual intracellular targets of purvalanol remain to be identified, and a method for the determination of its in vivo selectivity was developed. In this technique, cell extracts were screened for purvalanolinteracting proteins by affinity chromatography on immobilized inhibitor. In addition to CDK1, p42/p44 MAPK were found to be two major purvalanol-interacting proteins in five different mammalian cell lines (CCL39, PC12, HBL100, MCF-7 and Jurkat cells), suggesting the generality of the purvalanol/p42/p44 MAPK interaction. The Chinese hamster lung fibroblast cell line CCL39 was used as a model to investigate the anti-proliferative properties of purvalanol. The compound inhibited cell growth with a GI 50 value of 2.5 mM and induced a G2/M block when added to exponentially growing cells. It did not appear to trigger massive activation of caspase. We next tested whether CDKs and p42/p44 MAPK were actually targeted by the compound in vivo. p42/p44 MAPK activity was visualized using an Elk -Gal4 luciferase reporter system and CDK1 activity was detected by the phosphonucleolin level. When cells were treated with purvalanol, p42/p44 MAPK and CDK1 activities were inhibited in a dose-dependent manner. Furthermore, purvalanol inhibited the nuclear accumulation of p42/p44 MAPK, an event dependent on the catalytic activity of these kinases. We conclude that the anti-proliferative properties of purvalanol are mediated by inhibition of both p42/p44 MAPK and CDKs. These observations highlight the potency of moderate selectivity compounds and encourage the search for new therapeutics which simultaneously target distinct but relevant pathways of cell proliferation.

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The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism

Cited by 48 publications

References 63 publications

Crystal structure of human CDK4 in complex with a D-type cyclin

Crystal structure of human CDK4 in complex with a D-type cyclin

Understanding bistability in complex enzyme-driven reaction networks

p42/p44 MAPKs are intracellular targets of the CDK inhibitor purvalanol

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