Coccetti Paola, Zinzalla Vittoria, Tedeschi Gabriella, Russo Gian Luigi, Fantinato Sonia, Marin Oriano, Pinna Lorenzo A, Vanoni Marco, Alberghina Lilia
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi Milano-Bicocca, P.zza della Scienza 2, 20126 Milano, Italy.
Biochem Biophys Res Commun. 2006 Aug 4;346(3):786-93. doi: 10.1016/j.bbrc.2006.05.171. Epub 2006 Jun 6.
We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.
我们之前已确定酵母细胞周期蛋白依赖性激酶抑制剂Sic1的Ser201是蛋白激酶CK2的体外作用靶点。在此，我们通过使用特异性抗磷酸化Ser201抗体以及二维凝胶电泳结合基质辅助激光解吸电离质谱分析，提供了新的证据，表明在葡萄糖培养的细胞中，Sic1在其从头合成后不久的后期后期体内Ser201位点会发生磷酸化。从G1期细胞免疫纯化的Sic1中也检测到了这种磷酸化。与这些数据一致，我们还表明，在免疫纯化的Sic1复合物中检测到了CK2的催化α'亚基，其功能是细胞周期进程所必需的，并且在cka1δ cka2(ts)酵母菌株中，在非允许温度下CK2失活后，Ser201位点的磷酸化减少。这些数据有力地支持了CK2在体内使Sic1磷酸化的观点。
