Quantitative Reconstitution of Mitotic CDK1 Activation in Somatic Cell Extracts  Richard W. Deibler, Marc W. Kirschner  Molecular Cell  Volume 37, Issue 6, Pages 753-767 (March 2010) DOI: 10.1016/j.molcel.2010.02.023 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Overview of Cyclin-Induced Mitotic Entry (A) Schematic depicting experimental approach. See text for details. (B) Coomassie blue-stained polyacrylamide gels containing purified recombinant His6-cyclin B and His6-cyclin A. (C) HeLa S3 lysates (20 μg) prepared at the indicated times following G1/S synchronization (time = 0) were separated by SDS-PAGE and were immunoblotted with the shown antibodies (top). Recombinant cyclin A and B were added to quantify their concentration in the lysate. Shown is the quantification of three independent experiments with the error bars signifying ± 1 standard deviation (bottom). (D) Graphical representation of the DNA content of asynchronous cells and cells 5.5 hr after release from a double thymidine block as determined by FACS analysis. (E) Checkpoint status in somatic cell extracts. Cells were treated with 25 μM etoposide for 4 hr, lysed, and compared to the extracts used in the described in vitro system made with (NH4)2SO4 by immunoblotting. Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Biochemical Characterization of the Response of Somatic Cell Extracts to His6-Cyclin B (A) Autoradiogram of a dried gel containing radioactively phosphorylated histone H1 (top). His6-cyclin B was added to somatic cell extracts at the indicated concentrations and incubated for 20 min at room temperature. Kinase assays were performed to determine the amount of P32 incorporated into the CDK1 substrate histone H1. Quantification (n = 2–9) is shown (bottom) with error bars representing ± 1 standard deviation. The coefficient of variation was constant at 30%. (B) The effect of cyclin B on cellular proteins was analyzed by immunoblotting (phospho-CDK1, CDC25, and actin as a loading control) or by adding radiolabeled in vitro-translated proteins to the extract (WEE1 and securin). (C) Gel filtration of extracts following His6-cyclin B addition. Following a 20 min incubation at room temperature with the indicated cyclin B concentrations, extracts were loaded onto a Superdex 200 column, and fractions were analyzed by immunoblotting for CDK1, cyclin B, and WEE1. Shown are apparent molecular weight positions. (D) Cyclin B-CDK1 binding to mitotic regulators. His6-cyclin B bound to Ni-NTA agarose was added to the extract at a concentration below (100 nM) or above (2000 nM) the level for robust CDK1 activity. Following a 30 min incubation, the extract was centrifuged and the Ni-NTA beads were collected. The beads were washed three times with extract dialysis buffer, and the bound proteins were analyzed by SDS-PAGE and immunoblotting. Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 WEE1 and CDC25 Determine the Nature of the Response of the Extract to Cyclin B (A) Effect of WEE1 and CDC25 inhibition on CDK1 activity. Shown is an autoradiogram of a dried gel containing radioactively phosphorylated histone H1 (top) and quantification (bottom). Activities corresponding to stages 2 and 3 are also shown. Error bars correspond to ± 1 standard deviation (n = 3). (B) Effect of WEE1 (top) and CDC25 (bottom) inhibition on CDK1 Y15 phosphorylation. (C) Effect of WEE1 and CDC25 inhibition on CDK1 substrate phosphorylation. Shown is an immunoblot of CDC25C in the presence and absence of W2. (D) Effect of CDK1 inhibition on WEE1 phosphorylation (top) and CDK1 Y15 phosphorylation (bottom). Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 The Kinetics of the Extract Response (A) Autoradiogram of a dried polyacrylamide gel showing the time course of histone H1 kinase activation. Cell extract was mixed with 1000 or 100 nM cyclin B on ice and then incubated at room temperature. Aliquots were removed at the indicated time points and frozen in liquid nitrogen, and kinase activity was analyzed. (B) Immunoblot showing time course of CDK1 Y15 phosphorylation at the indicated concentrations of cyclin B. (C) Rate of binding of cyclin B to CDK1. Cyclin B bound to Ni-NTA was added to the extract at a concentration of 1000 nM and isolated at the indicated time points. Bound proteins were analyzed by immunoblotting for cyclin B and CDK1. Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Cyclin A Activation of CDKs (A) Activation of CDKs by His6-cyclin A. Recombinant His6-cyclin A was added at the indicated concentrations to the somatic cell extract and incubated for 20 min at room temperature. Histone H1 kinase activity was measured. Shown is an autoradiogram of a dried gel containing radiolabeled histone H1 (top) and the resulting (n = 3) quantification (bottom). Error bars represent ± 1 standard deviation. (B) Binding of CDK1 and CDK2 to cyclin A. BSA or 100 nM His6-cyclin A was added to the extracts. Ni-NTA agarose beads were added to both samples. The beads were washed three times in buffer. Shown are immunoblots for His6 (cyclin A), CDK1, and CDK2. (C) Immunodepletion of CDKs. Extracts were immunodepleted for CDK2 (bottom), CDK1 (middle), or mock depleted with normal rabbit IgG (top), and cyclin A was added. H1 kinase activity was measured, and an autoradiogram is shown. (D) CDK1 sets sensitivity to cyclin A. Buffer (top) or CDK1 (bottom) was added to extract along with cyclin A, and H1 kinase activity was measured. Shown is an autoradiogram. (E) Phosphorylation of CDK substrates by cyclin A-CDK complexes. WEE1 (top) or securin (bottom) were synthesized and labeled with S35 using in vitro translation. His6-cyclin A was added to the extracts at the indicated concentrations. After 20 min, the extracts were subject to SDS-PAGE and autoradiography. Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Cyclin A Enhances Cyclin B-CDK1 Activity (A) Representative autoradiogram showing cyclin B-dependent H1 kinase activity in the presence or absence of added 200 nM cyclin A (top). Graphical representation of CDK activity (n = 2) for extract containing cyclin A and B with error bars representing ± 1 standard deviation (bottom). (B) Shown is an immunoblot for CDK1 Y15 phosphorylation levels for extract containing buffer (top) or cyclin A (bottom) as a function of increasing cyclin B concentration. (C) The envelopes of nuclei were stained with rhodamine-conjugated DiI (pink) and the DNA with DAPI (blue). The nuclei were added to extract containing the indicated treatments at 30°C and imaged with a 60× objective 60 min later (top). The quantification of nuclear disintegration from three independent experiments (n = 50 per trial) is shown ± 1 standard deviation (below). Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Model of Mitotic Entry (A) Schematic showing the relationship between CDK1 activity and substrate phosphorylation. See text for details. (B) In G2, cyclin B accumulates throughout the cytoplasm and forms inactive complexes with CDK1. The concentration of cyclin B locally increases through localization at the centrosomes near the nucleus. This increased concentration activates cyclin B-CDK1 complexes. These complexes then can enter the nucleus. Cyclin A in the nucleus prevents WEE1 from inactivating these complexes. Molecular Cell 2010 37, 753-767DOI: (10.1016/j.molcel.2010.02.023) Copyright © 2010 Elsevier Inc. Terms and Conditions