Cdk1<sup>Clb2</sup> phosphorylates Esp1.

<p><b>(A</b>) Esp1 is phosphorylated <i>in vivo</i>. <i>ESP1</i>, <i>ESP1-myc13</i> and <i>pGAL-SWE1 ESP1-13myc</i> cells were grown in YEP + raffinose, arrested in mitosis with nocodazole and switched to YEP + galactose media to induce expression of Swe1. After 1 h, cells were washed in medium lacking phosphate, and grown for 30 min in the presence of [<sup>32</sup>P]orthophosphate. Esp1-13myc was immunoprecipitated with 9E10 antibody, run on a polyacrylamide gel and exposed to a phosphorimager screen or immunoblotted. (<b>B</b>) Esp1 contains six minimal Cdk1 consensus sites (S/TP): S13 and T16 (termed <i>N-terminal</i>), T1014, S1027 and T1034 (termed <i>central</i>) and S1280 (termed <i>C-terminal</i>). (<b>C</b>) Mutating Esp1 central residues prevents Esp1 phosphorylation <i>in vivo</i>. Left panels, <i>ESP1</i>, <i>esp1-2A</i>, <i>esp1-3A</i>, <i>esp1-1A</i>, <i>esp1-3A+2A</i>, <i>esp1-2A+1A</i>, <i>esp1-3A+1A</i> and <i>esp1-2A+3A+1A</i> cells were grown in YEP + dextrose, arrested in mitosis with nocodazole and labeled with [<sup>32</sup>P]orthophosphate as described in <b>(A)</b>. Right panels, wild-type, <i>cdc55Δ</i> and <i>rts1Δ</i> cells were grown in YEP + dextrose, arrested in G1 with α-factor and released into the cell cycle in the presence of nocodazole. After 90 min cells were labeled with [<sup>32</sup>P]orthophosphate as described in <b>(A).</b> Esp1 was immunoprecipitated with anti-Esp1 antibody, run on a polyacrylamide gel and exposed to a phosphorimager screen or immunoblotted. (<b>D</b>) Wild-type and <i>esp1-3A</i> cells were grown to log phase, arrested in G1 with α-factor, and released into the cell cycle (t = 0). α-factor was re-added at t = 60 min to arrest cells in the following G1. Samples were taken for immunoblotting at the indicated timepoints, and run on a polyacrylamide gel containing Phos-tag reagent (top panel), or a standard polyacrylamide gel (bottom panels) and immunoblotted with the indicated antibodies. Note that running and transferring of Phos-tag polyacrylamide gels is inconsistent and cell cycle-dependent changes in protein abundance observed in these panels may not accurately reflect changes in protein abundance. The Esp1 panels from the standard polyacrylamide gel more accurately reflect cell cycle changes in Esp1 abundance. (<b>E</b>) Cdk1<sup>Clb2</sup> phosphorylates the central region of Esp1 <i>in vitro</i>. Esp1 was immunoprecipitated from the strains in <b>(C)</b> growing asynchronously, incubated with γ-[<sup>32</sup>P]ATP and purified Cdk1<sup>Clb2-CBP</sup>, washed, run on a polyacrylamide gel, and exposed to a phosphorimager screen or immunoblotted with anti-Esp1 antibody. (<b>F</b>) PP2A<sup>Cdc55</sup> dephosphorylates Esp1 <i>in vitro</i>. Esp1 was immunoprecipitated from wild-type cells and phosphorylated with purified Cdk1<sup>Clb2-CBP</sup> and γ-[<sup>32</sup>P]ATP while immobilized on IgG-coupled magnetic beads. The beads were washed and incubated for the indicated times at room temperature with no addition (yellow lines), TAP-purified PP2A<sup>Cdc55</sup> (blue lines), or PP2A<sup>Cdc55</sup> and okadaic acid (OA) (red lines). The three reactions share a t = 0 sample that was taken before the additions. The dephosphorylation of Esp1 was quantified on a phosphorimager and the extent of dephosphorylation relative to t = 0 (average ± SEM) was graphed. The experiment shown is representative of one of three repeats.</p>