1. Kinase-Independent Function of Cyclin E
Yan Geng, Young-Mi Lee, Markus Welcker, Jherek Swanger, Agnieszka Zagozdzon, Joel D. Winer, James M. Roberts, Philipp Kaldis, Bruce E. Clurman, Piotr Sicinski 
Molecular Cell 
Volume 25, Issue 1, Pages (January 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Rescue of Cell-Cycle Reentry by Kinase-Deficient Cyclin E
(A and B) Wild-type (WT) or cyclin E-null (E1−/−E2−/−) MEFs were transduced with an empty vector (Vec) or with retroviruses encoding WT human cyclin E1 (WT E), cyclin E1 S75A mutant, or kinase-deficient cyclin E1 KD-E mutant (KD E). Cells were serum starved and then stimulated to reenter the cell cycle by serum addition. Incorporation of [3H]-thymidine was determined at the indicated time points.
(C) Mean thymidine incorporation at 21 hr after the stimulation in three independent experiments. Values observed in cyclin E-null cells transduced with wild-type cyclin E were set as 100%. Error bars denote the SD.
(D) Detection of endogenous mouse cyclin E and ectopically expressed human cyclin E in the transduced MEFs. Immunoblots were probed with an antibody recognizing both mouse and human cyclin E.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Analyses of Kinase-Deficient KD-E Cyclin E1 Mutant
(A) WT or cyclin E1−/−E2−/− MEFs were transduced with empty vector (Vec), or with retroviruses encoding human wild-type cyclin E1 (WT E), cyclin E1 S75A mutant, or kinase-deficient KD-E mutant (KD-E). Ectopically expressed cyclin E was immunoprecipitated with anti-human cyclin E1 antibody, and in vitro kinase reactions were performed with recombinant histone H1 or the retinoblastoma protein (pRB) as substrates.
(B) Similar in vitro kinase assays using recombinant CDC6, p107, and CDC25A proteins as substrates; human cyclin E1 or KD-E mutant was expressed in wild-type MEFs.
(C) Human H293 cells were transfected with Myc-tagged WT E or KD E. Cyclin E was immunoprecipitated with anti-Myc antibody, and in vitro kinase reactions were performed without adding any exogenous substrate. The strongest band (marked by a triangle) corresponds to autophosphorylated cyclin E. At least five cyclin E-associated proteins (marked by stars) become radioactively labeled in WT E immunoprecipitates. Molecular-weight standards (in kD) are indicated. Immunoprecipitates were probed with an anti-human cyclin E1 antibody (cyclin E) to ensure comparable amounts of immunoprecipitated WT and mutant cyclin E (bottom).
(D) MEFs derived from p27−/−p21−/− mice were transduced with retroviruses as described above. Human cyclin E was immunoprecipitated, and in vitro kinase reactions were performed as in (A). In addition, immunoprecipitates were blotted and probed with an anti-human cyclin E1 antibody (cyclin E) to ensure comparable amounts of immunoprecipitated WT and mutant cyclin E.
(E) WT MEFs were transduced with retroviruses encoding human cyclin E, as above. Human cyclin E was immunoprecipitated, and the immunoblots were probed with an antibody against phosphoserine 384 residue of cyclin E (Ep). Immunoblots were also probed with anti-human cyclin E antibody (cyclin E) to ensure comparable amounts of immunoprecipitated WT and mutant cyclin E. A nonspecific band, also present in cells transduced with empty vector (Vec), i.e., cells not expressing human cyclin E, is marked by an asterisk.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Rescue of Oncogene Resistance by Kinase-Deficient Cyclin E
(A) Appearance of monolayers of MEFs transduced with retroviruses encoding Ras plus dominant-negative p53.
(B) Mean number of oncogenic foci in three independent experiments. Values for cyclin E-null cells transduced with wild-type cyclin E were set as 100%. Error bars denote the SD.
(C) Expression levels of Ras in nontransduced and in transduced (+ Ras/DNp53) cells were determined by western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Loading of MCM and Cyclin E onto Chromatin
(A) Cyclin E-null (E1−/−E2−/−) MEFs were transduced with retroviruses encoding WT E, KD E, or empty vectors (Vec). Cells were serum starved, stimulated to reenter the cell cycle by serum addition, and collected at the indicated time points. The incorporation of MCM2 into the chromatin-bound fraction and its levels in the “free” fraction were determined by western blotting. WT cells are shown for reference.
(B) Similar analyses of incorporation of ectopically expressed human cyclin E into chromatin-bound fraction in cyclin E1−/−E2−/− MEFs.
(C) WT MEFs were serum starved and then stimulated to reenter the cell cycle as above. At the indicated time points, the incorporation of the endogenous cyclin E1, CDC6, MCM2, and ORC2 into the chromatin-bound fraction and their levels in the “free” fraction were determined by western blotting.
(D) Chromatin-bound fraction was digested with DNase I, and the presence of cyclin E and MCM2 in the supernatant (sup) and pellet was determined by western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Localization of Cyclin E on Chromatin
(A) WT MEFs were subjected to in situ extraction. The nonextractable (chromatin-bound) fraction was stained with DAPI for DNA or with anti-MCM2 or anti-cyclin E1 antibodies, and the images were analyzed by confocal microscopy. Merge shows superimposed anti-MCM2 and anti-cyclin E staining. Inset, higher magnification of the selected nuclei. The bottom panels show staining of cells that underwent digestion with DNase (+DNase) to remove DNA-bound proteins.
(B) Similar analyses of human HeLa cells subjected to in situ extraction. Inset, higher magnification of the selected nuclei. For comparison, nuclei were stained for MCM2 and PCNA (bottom).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Physical Interaction of Cyclin E with MCM and with CDT1
(A) Normal loading of CDT1 during G0 → S phase progression of cyclin E null cells. WT or cyclin E-null (E1−/−E2−/−) MEFs were rendered quiescent by serum deprivation and then stimulated to reenter the cell cycle by serum readdition. Cells were collected at the indicated time points, and the incorporation of CDT1 into the chromatin-bound fraction and its levels in the free fraction were determined by western blotting.
(B) Coimmunoprecipitation of cyclin E1 with endogenous MCMs. H293 cells were transfected with WT human cyclin E1 (H293 + WT E). Cyclin E1 was immunoprecipitated (E-IP) from these cells or from intact H293 cells (H293), and the immunoblots were probed with antibodies against MCM2 or MCM7. As a negative control, lysates were immunoprecipitated with preimmune serum (contr). Straight lysates (lysate) were run and imunoblotted in parallel.
(C) H293 cells were transfected with Myc epitope-tagged wild-type cyclin E1 (H293 + WT E) or kinase-deficient KD-E mutant (H293 + KD E). Cyclin E1 was immunoprecipitated (E-IP) from these cells with anti-Myc tag antibody, and the immunoblots were probed with antibodies against MCM2 or MCM7. Controls were as in (B).
(D) Bacterially expressed GST-MCM2 or GST-MCM7 proteins were purified by using glutathione Sepharose and incubated with extracts of H293 cells. Bound proteins were resolved on PAGE gels, immunoblotted, and probed with an anti-cyclin E1 antibody. As a negative control, lysates were incubated with beads containing GST only (GST). Straight lysates (lysate) were run and immunoblotted in parallel.
(E) WT E or KD E was in vitro translated with wheat germ lysate, labeled, and incubated with GST-MCM2, GST-MCM7, or GST alone (negative control). Bound proteins were resolved on PAGE gels and visualized by autoradiography. Ten percent of radiolabeled cyclin E input (“input”) was run in parallel.
(F) Coimmunoprecipitation of cyclin E1 with endogenous CDT1. H293 cells were transfected with wild-type cyclin E1 (H293 + WT E). Cyclin E1 was immunoprecipitated (E-IP) from these cells or from intact H293 cells (H293), and the immunoblots were probed with an antibody against CDT1. Controls were as in (B).
(G) Bacterially expressed GST-CDT1 protein was purified by using glutathione Sepharose and incubated with extracts of H293 cells. Bound proteins were resolved on PAGE gels, immunoblotted, and probed with an anti-cyclin E1 antibody. Controls were as in (D).
(H) WT E or KD E was in vitro translated with wheat germ lysate, labeled, and incubated with GST-CDT1 or GST alone (negative control). Bound proteins were resolved on PAGE gels and visualized by autoradiography. Ten percent of radiolabeled cyclin E input (“input”) was run in parallel.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 Cyclin E Function in G0 → S Phase Progression
A model integrating various kinase-dependent functions with a unique kinase-independent function of cyclin E (depicted in red) in assembly of prereplication complexes during G0 → S phase progression. The catalytic action of cyclin E-CDK complexes is depicted by thick arrows.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions