Pmk1 kinase-dependent phosphosite mapping on Slp1

In late mitosis, the timely polyubiquitylation and subsequent degradation of securin and cyclin B by anaphase-promoting complex/cyclosome (APC/C) play a critical role for anaphase onset and chromosome segregation. Binding of Cdc20, the co-activator of the APC/C, is critical for activation of APC/C at anaphase entry. We found that one MAPK, Pmk1, is involved in phosphorylation of Slp1Cdc20 (the Cdc20 homologue in the fission yeast Schizosaccharomyces pombe), which may promote its ubiquitylation and subsequent degradation, and thus impede APC/C activation and anaphase entry. To gain a full picture of Pmk1-dependent phosphosite map of Slp1Cdc20, we purified the APC/C-associated Slp1Cdc20 by immunoprecipitation of one APC/C subunit Apc15 in metaphase-arrested nda3-km311 cells. Apc15-GFP was purified from wild type, pek1DD [i.e. pek1(S234D,T238D)] and pmk1∆ cells respectively. Samples were separated by SDS-PAGE, and Coomassie blue-stained gel slices were excised and submitted for mass spectrometry analyses.

在有丝分裂晚期，后期促进复合物/周期体（anaphase-promoting complex/cyclosome, APC/C）对分离酶抑制蛋白（securin）和细胞周期蛋白B（cyclin B）的适时多泛素化及其后续降解，对于后期起始与染色体分离至关重要。APC/C的共激活因子细胞分裂周期蛋白20（Cdc20）的结合，是APC/C在后期进入时激活的关键前提。我们发现，丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）家族成员Pmk1参与了Slp1^Cdc20——粟酒裂殖酵母（Schizosaccharomyces pombe）中的Cdc20同源物——的磷酸化修饰，该过程可能促进其泛素化与后续降解，进而阻碍APC/C的激活及后期进入。为完整绘制Pmk1依赖的Slp1^Cdc20磷酸化位点图谱，我们在中期阻滞的nda3-km311细胞中，通过免疫沉淀APC/C亚基Apc15，纯化得到了与APC/C结合的Slp1^Cdc20。分别从野生型、pek1DD[即pek1(S234D,T238D)]以及pmk1Δ细胞中纯化获得Apc15-GFP。样品经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，切取考马斯亮蓝染色的凝胶条带，进行质谱分析。