Volume 1, Issue 3, Pages 371-380 (February 1998) PKA and MPF-Activated Polo-like Kinase Regulate Anaphase-Promoting Complex Activity and Mitosis Progression  Shuji Kotani, Stuart Tugendreich, Mika Fujii, Pia-Marie Jorgensen, Nobumoto Watanabe, Christer Hoog, Philip Hieter, Kazuo Todokoro  Molecular Cell  Volume 1, Issue 3, Pages 371-380 (February 1998) DOI: 10.1016/S1097-2765(00)80037-4

Figure 1 The Binding and Phosphorylation of Cdc16, Cdc27, and Tsg24 with Plk (A) The immunoprecipitates with anti-Plk antibody of NIH3T3 cells in G1/S phase or prometaphase were incubated with [γ-32P]ATP. The total reaction mix in G1/S boundary (lane 1) or mitosis (lane 2) was applied onto SDS-PAGE and autogradiographed. After boiling the reaction mix with SDS, the phosphorylated proteins were reimmunoprecipitated with anti-Plk- (lane 3), anti-Cdc16- (lane 4) and anti-Cdc27 (lane 5)-specific antibodies and resolved by SDS-PAGE. Arrows indicate the phosphorylated Plk, Cdc16, and Cdc27. The phosphorylated Tsg24, indicated by an arrow, was identified by immunoblot analysis (not shown, but see Figure 3). (B) The electrophoretic mobilities of mouse Cdc16, Cdc27, and Tsg24 during the cell cycle. NIH/3T3 cell extracts arrested at G1/S phase (lane 1) or prometaphase (lanes 2 and 3) were immunoblotted with anti-Tsg24 (upper panel), anti-Cdc27 (middle panel), or anti-Cdc16 (lower panel) antibody before (lanes 1 and 2) and after (lane 3) λ protein phosphatase treatment. Numbers at left indicate the molecular weights of the bands in kilodaltons. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 3 MPF-Activated Plk and PKA Phosphorylate Purified APC (A) MPF-activated Plk phosphorylates purified APC. The purified mouse APC was incubated with [γ-32P]ATP with (+) or without (−) recombinant MPF, His-Plk, and butyrolactone I. Asterisks indicate that MPF and His-Plk were preincubated before mixing with butyrolactone I. Lanes 1–6 show SDS-PAGE of the total reaction mix. Lanes 7 and 8 show SDS-PAGE of the reimmunoprecipitates of the reaction mix with anti-Cdc16 (lane 7) and anti-Cdc27 (lane 8) antibody following boiling with SDS. Lanes 9–11 show immunoblot analysis of the phosphorylated APC with anti-Cdc16 (lane 9), anti-Cdc27 (lane 10), and anti-Tsg24 (lane 11) antibody. (B) PKA phosphorylates purified APC. The purified mouse APC was incubated with [γ-32P]ATP with bovine PKA catalytic subunit. Lane 1 shows SDS-PAGE of the total reaction mix. Lane 2 shows SDS-PAGE of the reimmunoprecipitates of the reaction mix with anti-Cdc27 antibody. The immunoblot analysis of the reaction mix was performed with anti-Cdc27 (lane 3) or anti-Tsg24 antibody (lane 4). The phosphorylated Cdc27 and the supershifted Tsg24 are indicated by arrows. (C) Immunoblots of the purified APC proteins at S phase (without kinase reaction) with anti-Cdc16 (lane 1), Cdc27 (lane 2), and Tsg24 (lane 3) antibodies. (D) Immunoblots of Tsg24 in the purified APC before (lane 1) and after Plk (lane 2) or PKA (lane 3) treatment. An arrow indicates the supershifted Tsg24. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 2 In Vitro Phosphorylation of the Recombinant APC Subunits Cdc16, Cdc27, and Tsg24 by Recombinant Kinases MPF, Plk, and PKA (A) In vitro Plk assay with or without MPF. Human His-Cdc16 (lanes 5–10), human His-Cdc27 (lanes 11–16), mouse His-Tsg24-N and His-Tsg24-C (lanes 17–28), which were all produced in bacteria, were phosphorylated by mouse His-Plk in the presence or absence of Cdc2-GST-cyclin B complex purified from the baculovirus system and MPF inhibitor butyrolactone I. Samples were mixed in various combinations as indicated: (+) presence; (−) absence. Lanes 1–4 show the kinase reactions without APC substrates. The samples were resolved in SDS-PAGE and visualized by BAS2000. Asterisks indicate that MPF and His-Plk were preincubated before mixing with butyrolactone I. Arrows indicate the position of phosphorylated His-Plk, GST-cyclin B, His-Cdc16, His-Cdc27, or His-Tsg24-N. Two human phosphorylated Cdc27 were detected at 99 and 125 kDa. A band of about 72 kDa, which was always seen above His-Plk, is a contaminated bacterial protein which can be phosphorylated by His-Plk. (B) In vitro Plk assay after MPF-activated Plk was repurified. Lane 1 shows the total reaction mix of His-Plk, MPF, and [γ-32P]ATP. Lane 2 shows the MPF-activated His-Plk, which was repurified by His-Bind column with 0.5 M NaCl washing. The total reaction mix of the repurified MPF-activated His-Plk, [γ-32P]ATP, and His-Cdc16 is shown in lane 3. The reaction mix for His-Cdc27 is shown in lane 4, for His-Tsg24-N in lane 5, and for His-Tsg24-C in lane 6. (C) The electrophoretic mobilities of human Cdc16 and Cdc27 during the cell cycle. HeLa cell extracts treated with aphidicolin (lane 1) or nocodazole (lane 2) were immunoblotted with anti-Cdc27 (upper panel) or anti-Cdc16 (lower panel) antibody. The numbers at left indicate the molecular weights of the proteins in kilodaltons. (D) In vitro PKA assay with the recombinant His-tagged APC subunits. The bovine PKA catalytic subunit (cAMP independently active form) was incubated with the purified recombinant His-tagged APC subunits. Samples were mixed in various combinations as indicated: (+) presence; (−) absence. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 4 MPF-Activated Plk Activates APC, but PKA Inactivates APC The purified APC in S phase was incubated with (+) or without (−) His-Plk, MPF, butyrolactone I, and PKA, and the activity to ubiquitinate GST-cyclin B was measured in the presence of recombinant E1 and E2-C (lanes 1–8). The samples were applied onto SDS-PAGE and the polyubiquitinated GST-cyclin B was detected by immunoblotting with anti-cyclin B antibody. Asterisks indicate that MPF and His-Plk were preincubated prior to being mixed with butyrolactone I (lane 6). Lane 9 shows the APC activity after preincubation of APC with MPF-activated Plk, which induced full APC activity, followed by incubation with PKA. Lane 10 shows the APC activity after preincubation of APC with PKA followed by incubation with MPF-activated Plk. The number (instead of + or −) indicates the order of addition of the kinases. A number of polyubiquitinated GST-cyclin B bands are shown as Ub-cyclin B. Arrow indicates the position of nonubiquitinated GST-cyclin B. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 5 Both PKA and Plk Activities Are High during Mitosis, but PKA Activity Falls before Plk Activity Reaches the Maximum Level The maximum activity is defined as 1, and the relative activities are plotted against time (h). (A) The synchrony of the cells. NIH/3T3 cells were double-blocked at the G1/S boundary with aphidicolin and released. The synchrony of the cells was monitored by mitotic index (bars) and by Cdc2 kinase assay (open circles). (B) Specific Plk activity (squares) and PKA activity (closed circles) during cell cycle. The immunoprecipitates with anti-Plk antibody at the indicated time points were incubated in a kinase buffer containing His-Cdc16 as a specific substrate and [γ-32P]ATP. The samples were resolved by 7% SDS-PAGE, and the radioactivity of the labeled substrates was counted. PKA activities in the total cell extracts at the indicated time points were measured using fluorescent kemptide as a specific substrate and quantified. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 6 Changes in Plk Activity, PKA Activity, Phosphorylation Status of APC Subunits, and APC Activity after Prometaphase The maximal level was defined as one, and the relative values are plotted against time (min) in (A), (B), and (D). (A) The synchrony of the cells. NIH/3T3 cells were arrested in prometaphase with nocodazole, and loosely attached mitotic cells were harvested. The synchrony of the cells was monitored by mitotic index (bars), by Cdc2 kinase assay (open circles), and by anaphase index (triangles). (B) Specific Plk activity (squares) and PKA activity (closed circles) after prometaphase. Plk and PKA activities were measured at the indicated time points after prometaphase. (C) Phosphorylation status and the level of Tsg24, Cdc27, Cdc16, cyclin B, and Erk1 after prometaphase. The cell extracts at the indicated time points were immunoblotted with each specific antibody. Erk1 was used as an internal control that is constantly expressed during the cell cycle. Arrows indicate the phosphorylated APC subunits. (D) APC activity after prometaphase. The cell extracts were incubated with GST-cyclin B and the polyubiquitinated GST-cyclin B was detected by anti-cyclin B antibody. Polyubiquitinated GST-cyclin B bands are shown as Ub-cyclin B. An arrow indicates the position of nonubiquitinated GST-cyclin B. The lower panel shows the changes of APC activity after prometaphase. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)

Figure 7 Plk Activates APC, and PKA Suppresses APC Activation by Plk In Vivo (A) Phosphorylated Cdc16, Cdc27, and Tsg24 observed in Plk-expressing NIH/3T3 cells. The cell extracts of mock-transfected NIH/3T3 cells (lane 1), DN-Plk-expressing cells (lane 2), Plk-expressing cells (lane 3), CPT-cAMP-treated cells (lane 4), and CPT-cAMP-treated Plk-expressing cells (lane 5) were immunoblotted with anti-Tsg24 (upper), anti-Cdc27- (middle panel), or anti-Cdc16-specific (lower panel) antibody. (B) APC activity detected in Plk-expressing cells is suppressed by PKA. GST-cyclin B was incubated with the same cell extracts used for the immunoblots shown in (A). Samples were applied to SDS-PAGE and immunoblotted with anti-cyclin B antibody. An arrow indicates nonubiquitinated intact GST-cyclin B. Molecular Cell 1998 1, 371-380DOI: (10.1016/S1097-2765(00)80037-4)