Limits...
Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.

Furstenthal L, Kaiser BK, Swanson C, Jackson PK - J. Cell Biol. (2001)

Bottom Line: In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis.In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin.Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stangford University School of Medicine, Palo Alto, California 94305, USA.

ABSTRACT
Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

Show MeSH

Related in: MedlinePlus

Specific mitotic kinases are capable of phosphorylating cyclin E and displacing cyclin E–Cdk2 from chromatin; Cdc14 can oppose phosphorylation by these kinases. (A) Sperm chromatin assembled in interphase LSS (in the presence of cycloheximide) for 1 h was subsequently treated with buffer (lane 1), 1 U of MAP kinase (lane 2), cyclin B–Cdc2 (lane 3), Plk1 (lane 4), or 10 μM okadaic acid (lane 5) for 10 min. Chromatin was extracted, and associated proteins were resolved by SDS-PAGE and Western blotted for the presence of cyclin E or ORC. (B) Purified GST–Xcylin E was incubated with buffer (lanes 1 and 2), MAP kinase (lanes 3 and 4), cyclin B–Cdc2 (lanes 5 and 6), Plk1 (lanes 7 and 8), or cyclin E–Cdk2 (lane 9–10) in the presence of γ[32P]ATP. After 30 min, 2 μM GST–Cdc14 was added to indicated samples (lanes 2, 4, 6, 8, and 10), and all samples were incubated for an additional 30 min. Reactions were resolved by SDS-PAGE, and phosphorylated GST–cyclin E was visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199215&req=5

Figure 8: Specific mitotic kinases are capable of phosphorylating cyclin E and displacing cyclin E–Cdk2 from chromatin; Cdc14 can oppose phosphorylation by these kinases. (A) Sperm chromatin assembled in interphase LSS (in the presence of cycloheximide) for 1 h was subsequently treated with buffer (lane 1), 1 U of MAP kinase (lane 2), cyclin B–Cdc2 (lane 3), Plk1 (lane 4), or 10 μM okadaic acid (lane 5) for 10 min. Chromatin was extracted, and associated proteins were resolved by SDS-PAGE and Western blotted for the presence of cyclin E or ORC. (B) Purified GST–Xcylin E was incubated with buffer (lanes 1 and 2), MAP kinase (lanes 3 and 4), cyclin B–Cdc2 (lanes 5 and 6), Plk1 (lanes 7 and 8), or cyclin E–Cdk2 (lane 9–10) in the presence of γ[32P]ATP. After 30 min, 2 μM GST–Cdc14 was added to indicated samples (lanes 2, 4, 6, 8, and 10), and all samples were incubated for an additional 30 min. Reactions were resolved by SDS-PAGE, and phosphorylated GST–cyclin E was visualized by autoradiography.

Mentions: To determine if any of several essential mitotic kinases were capable of phosphorylating cyclin E and displacing the cyclin E–Cdk2 complex from chromatin, we treated chromatin assembled in interphase LSS extracts with cyclin B–Cdc2, MAP kinase, or the polo-like kinase (Plk1) (Murray and Kirschner 1989; Lane and Nigg 1996; Guadagno and Ferrell 1998) and isolated assembled chromatin. Although treatment with Plk1 had no effect, cyclin B–Cdc2 efficiently removed cyclin E–Cdk2 from chromatin (Fig. 8 A). Addition of MAP kinase could also displace the majority of cyclin E–Cdk2 from chromatin, but less efficiently (Fig. 8 A). Both cyclin B–Cdc2 and MAP kinase phosphorylated purified GST–cyclin E in vitro (Fig. 8 B), suggesting that the effect on cyclin E may be direct. Plk1 also phosphorylated GST–cyclin E in vitro (Fig. 8 B), but the significance of this remains unclear. The Cdc14 phosphatase was capable of reversing the phosphorylation of cyclin E by both Cdc2 and MAP kinase but not by Plk1 (Fig. 8 B), indicating that Plk1 likely phosphorylates cyclin E on different sites from Cdc2 and MAP kinase.


Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.

Furstenthal L, Kaiser BK, Swanson C, Jackson PK - J. Cell Biol. (2001)

Specific mitotic kinases are capable of phosphorylating cyclin E and displacing cyclin E–Cdk2 from chromatin; Cdc14 can oppose phosphorylation by these kinases. (A) Sperm chromatin assembled in interphase LSS (in the presence of cycloheximide) for 1 h was subsequently treated with buffer (lane 1), 1 U of MAP kinase (lane 2), cyclin B–Cdc2 (lane 3), Plk1 (lane 4), or 10 μM okadaic acid (lane 5) for 10 min. Chromatin was extracted, and associated proteins were resolved by SDS-PAGE and Western blotted for the presence of cyclin E or ORC. (B) Purified GST–Xcylin E was incubated with buffer (lanes 1 and 2), MAP kinase (lanes 3 and 4), cyclin B–Cdc2 (lanes 5 and 6), Plk1 (lanes 7 and 8), or cyclin E–Cdk2 (lane 9–10) in the presence of γ[32P]ATP. After 30 min, 2 μM GST–Cdc14 was added to indicated samples (lanes 2, 4, 6, 8, and 10), and all samples were incubated for an additional 30 min. Reactions were resolved by SDS-PAGE, and phosphorylated GST–cyclin E was visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199215&req=5

Figure 8: Specific mitotic kinases are capable of phosphorylating cyclin E and displacing cyclin E–Cdk2 from chromatin; Cdc14 can oppose phosphorylation by these kinases. (A) Sperm chromatin assembled in interphase LSS (in the presence of cycloheximide) for 1 h was subsequently treated with buffer (lane 1), 1 U of MAP kinase (lane 2), cyclin B–Cdc2 (lane 3), Plk1 (lane 4), or 10 μM okadaic acid (lane 5) for 10 min. Chromatin was extracted, and associated proteins were resolved by SDS-PAGE and Western blotted for the presence of cyclin E or ORC. (B) Purified GST–Xcylin E was incubated with buffer (lanes 1 and 2), MAP kinase (lanes 3 and 4), cyclin B–Cdc2 (lanes 5 and 6), Plk1 (lanes 7 and 8), or cyclin E–Cdk2 (lane 9–10) in the presence of γ[32P]ATP. After 30 min, 2 μM GST–Cdc14 was added to indicated samples (lanes 2, 4, 6, 8, and 10), and all samples were incubated for an additional 30 min. Reactions were resolved by SDS-PAGE, and phosphorylated GST–cyclin E was visualized by autoradiography.
Mentions: To determine if any of several essential mitotic kinases were capable of phosphorylating cyclin E and displacing the cyclin E–Cdk2 complex from chromatin, we treated chromatin assembled in interphase LSS extracts with cyclin B–Cdc2, MAP kinase, or the polo-like kinase (Plk1) (Murray and Kirschner 1989; Lane and Nigg 1996; Guadagno and Ferrell 1998) and isolated assembled chromatin. Although treatment with Plk1 had no effect, cyclin B–Cdc2 efficiently removed cyclin E–Cdk2 from chromatin (Fig. 8 A). Addition of MAP kinase could also displace the majority of cyclin E–Cdk2 from chromatin, but less efficiently (Fig. 8 A). Both cyclin B–Cdc2 and MAP kinase phosphorylated purified GST–cyclin E in vitro (Fig. 8 B), suggesting that the effect on cyclin E may be direct. Plk1 also phosphorylated GST–cyclin E in vitro (Fig. 8 B), but the significance of this remains unclear. The Cdc14 phosphatase was capable of reversing the phosphorylation of cyclin E by both Cdc2 and MAP kinase but not by Plk1 (Fig. 8 B), indicating that Plk1 likely phosphorylates cyclin E on different sites from Cdc2 and MAP kinase.

Bottom Line: In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis.In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin.Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stangford University School of Medicine, Palo Alto, California 94305, USA.

ABSTRACT
Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

Show MeSH
Related in: MedlinePlus