Resnick R J, Taylor S J, Lin Q, Shalloway D
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Oncogene. 1997 Sep;15(11):1247-53. doi: 10.1038/sj.onc.1201289.
Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein which associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. We now show that Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues exclusively during mitosis as well as on serine residues during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, was phosphorylated on threonine and serine residues after incubation with mitotic lysates several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis.
Sam68（有丝分裂相关的Src结合蛋白）是一种含有SH3（Src同源结构域3）、SH2（Src同源结构域2）和RNA结合结构域的蛋白质，它以有丝分裂特异性方式与c-Src的野生型和激活形式结合并被其酪氨酸磷酸化。我们现在发现，从HeLa S3或NIH3T3细胞中免疫沉淀得到的Sam68仅在有丝分裂期间苏氨酸残基被磷酸化，而在间期和有丝分裂期间丝氨酸残基均被磷酸化。以谷胱甘肽S-转移酶（GST）融合蛋白形式表达的重组Sam68，与有丝分裂裂解物孵育后，其苏氨酸和丝氨酸残基的磷酸化程度比与未同步化的裂解物孵育后高几倍。通过以下方法确定Cdc2是有丝分裂苏氨酸磷酸化的激酶：（1）用抗Cdc2抗体从细胞裂解物中免疫去除有丝分裂Sam68激酶；（2）细胞周期蛋白依赖性激酶抑制剂olomoucine在体外和体内抑制Sam68磷酸化；（3）用纯化的Cdc2使Sam68磷酸化。这些数据表明Sam68是Cdc2的直接靶标，因此可能在有丝分裂期间介导其一些生物学效应。
