Nuclear accumulation of cyclin E/Cdk2 triggers a concentration-dependent switch for the destruction of p27Xic1 - PubMed (original) (raw)

Nuclear accumulation of cyclin E/Cdk2 triggers a concentration-dependent switch for the destruction of p27Xic1

C Swanson et al. Proc Natl Acad Sci U S A. 2000.

Abstract

The action of cyclin-dependent kinases (CDKs) is regulated by phosphorylation, cyclin levels, the abundance of CDK inhibitors, and, as recently has been shown for cyclin B/cdc2, their localization. It is unclear how localization regulates the action of cyclin E/Cdk2 and its inhibitors. Here, we show that the closest known Xenopus laevis homolog of mammalian Cdk2 inhibitors p27(Kip1) and p21(CIP1), Xic1, is concentrated, ubiquitinated, and destroyed in the nucleus. Furthermore, Xic1 destruction requires nuclear import, but not nuclear export, and requires the formation of a transport-competent nuclear envelope, but not interactions between the lamina and chromatin. We show that (i) cyclin E/Cdk2 and Xic1 are transported into the nucleus as a complex and that Xic1 destruction requires the activity of cyclin E, (ii) that phosphorylation of Xic1 by cyclin E/Cdk2 bypasses the requirement for nuclear formation, and (iii) that the phosphorylation of Xic1 by cyclin E/Cdk2 is concentration dependent and likely realized through second-order interactions between stable cyclin E/Cdk2/Xic1 ternary complexes. Based on these results we propose a model wherein nuclear accumulation of the cyclin E/Cdk2/Xic1 complex triggers a concentration-dependent switch that promotes the phosphorylation of Xic1 and, consequently, its ubiquitination and destruction, thus allowing subsequent activation of cyclin E/Cdk2.

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Figures

Figure 1

Figure 1

Xic1 is rapidly transported into and ubiquitinated in the nucleus. (A) Destruction of Xic1 requires formation of nuclei. 35S-labeled IVT Xic1 was added to the indicated extract fraction(s) plus or minus sperm DNA. Reactions were processed and analyzed as described in Materials and Methods. Light microscopy confirmed that nuclei formed only in S-phase extract and in the reconstituted cytosolic and membrane fractions (Cyt and Mem). (B) Xic1 is ubiquitinated in the nucleus. Reactions were prepared as in A with sperm DNA, separated into the cytoplasmic and nuclear fractions at the indicated times, and processed as described in Materials and Methods. Samples were analyzed by PhosphorImaging or Western blotting with anti-cyclin E antibody. Subtypes of Xic1 are indicated: Xic1Cyt is the cytoplasmic fraction, Xic1-Ub0 is the unubiquitinated nuclear fraction, and Xic1-Ubn is the ubiquitinated nuclear fraction. The amount of added IVT Xic1 did not measurably affect the normal time course of DNA replication. (C) Xic1 destruction begins rapidly in preformed nuclei. Sperm and energy were mixed with interphase extract and incubated for 50 min to allow nuclei to form. After nuclear formation was confirmed by microscopy, IVT Xic1 and additional extract were added to the reaction (t = 0 min). Samples were removed at indicated times and processed as in B. (D) The modified forms of Xic1 are ubiquitinated. Reactions were prepared as in A and ubiquitin (Ub) or methylated ubiquitin (MeUb) were added and processed at indicated times as in B. To assess the overall effect of MeUb on destruction, comparison of the summed amount of cytoplasmic (Cyt) and nuclear (Nuc) Xic1 remaining was quantitated to be more than 7-fold greater in the sample with added MeUb.

Figure 2

Figure 2

Xic1 is destroyed inside the nucleus in a cyclin E-dependent manner. (A) Cyclin E and Xic1 form a complex in extract. IVT Xic1 was added to extract. After 30 min the reactions were stopped by dilution into buffer with preimmune or anti-cyclin E sera. After 1 h protein A-Sepharose beads were added. After 20 min the beads were isolated and washed. Load (L), supernatant (S), and pellet (P) fractions were resolved by SDS/PAGE. (B) Imunodepletion of cyclin E blocks ubiquitination and destruction but not nuclear accumulation of Xic1. Mock or anti-cyclin E-depleted (3) extracts were assayed for Xic1 transport and destruction as in Fig. 1. (C) The addition of 1 μM LMB does not alter the kinetics of the nuclear accumulation, ubiquitination, and destruction of Xic1.

Figure 3

Figure 3

Disruption of the nuclear lamina does not block Xic1 destruction. Human LAP2 fragments 1–408 and 1–187 (see Results) were added to Xic1 destruction and replication assays (see Materials and Methods). Destruction activity was defined as the fraction of Xic1 degraded as determined by quantification on the PhosphorImager. Values were normalized to the unperturbed samples.

Figure 4

Figure 4

Prephosphorylation of Xic1 on Thr-205 by cyclin E/Cdk2 bypasses the nuclear requirement for Xic1 destruction. (A) Prephosphorylated Xic1 is destroyed even in the absence of nuclei. GST-Xic1 was incubated with baculovirus-purified cyclin E/Cdk2 in the presence (lower tier) or absence of ATP (upper tier) for 30 min. Destruction assays were conducted plus or minus sperm and MG132 as indicated. Reactions were processed as in Fig. 1_A_ except that membranes were immunoblotted with anti-Xic1 antibody. (B) Cyclin E/Cdk2 phosphorylates Xic1 on Thr-205. Phosphorylated (III and IV) and unphosphorylated (I and II) GST-Xic1 were prepared by kinase reactions with or without ATP (see Materials and Methods). Samples were digested with trypsin and analyzed by matrix-assisted laser desorption ionization–time of flight MS.

Figure 5

Figure 5

Phosphorylation of Xic1 by cyclin E is second order with respect to concentration. (A) Schematic models describing mechanisms by which cyclin E/Cdk2 might phosphorylate Xic1. Mechanism 1 is an intracomplex interaction (first order), whereas mechanisms 2 and 3 are intercomplex interactions (second order). (B) Cyclin E/Cdk2 and MBP-Xic1 were mixed in equimolar amounts. After 30 min at room temperature, the reaction was diluted to the indicated concentrations, and kinase reactions were initiated, processed, and analyzed as described in Materials and Methods. Values for Xic1 phosphorylation were normalized by concentration and fit to a linear curve (_R_2 = 0.99). (C) Varying concentrations of MBP-Xic1 were incubated with a fixed concentration of cyclin E/Cdk2 (400 nM). After 30 min the Xic1 phosphorylation reactions were initiated, processed, and analyzed as in A except that the counts were plotted versus the concentration squared through and fit to a linear curve (_R_2 = 0.97).

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