Mahony D, Parry D A, Lees E
Department of Cell Signaling, DNAX Research Institute, Palo Alto, California 94304, USA.
Oncogene. 1998 Feb 5;16(5):603-11. doi: 10.1038/sj.onc.1201570.
We have performed biochemical analyses of cdk6 complexes in T cells. By gel filtration chromatography we observed at least three cdk6 containing complexes in the cell, the most abundant eluting at 450 kDa and 50-70 kDa and a minor complex eluting at 170 kDa. Cyclin D was present in the minor 170 kDa complex which co-eluted with the peak of cdk associated in vitro Rb kinase activity. Analysis of proteins that co-immunoprecipitated with cdk6 showed that the 450 kDa complex contained both Hsp90 and CDC37. The 50-70 kDa complex was made up of two moieties, a 66 kDa complex containing cdk6 bound to p19INK4d and monomeric cdk6. The subcellular localisation of the cdk6 complexes was analysed by preparing cytoplasmic and nuclear extracts. The 450 kDa complex was shown to be predominantly cytoplasmic, whereas the 170 kDa cyclin D/cdk6 and the 50-70 kDa complexes were present in both nuclear and cytoplasmic compartments. When these same extracts were assayed for cdk6 associated kinase activity, only the nuclear cdk6 complexes were active. These data suggest that even though there are cdk6/cyclin D complexes detectable in both the cytoplasm and nucleus, only the cdk6 that is in the nucleus is active.
我们对T细胞中的cdk6复合物进行了生化分析。通过凝胶过滤色谱法，我们在细胞中观察到至少三种含有cdk6的复合物，最丰富的在450 kDa和50 - 70 kDa处洗脱，还有一种较小的复合物在170 kDa处洗脱。细胞周期蛋白D存在于较小的170 kDa复合物中，该复合物与体外cdk相关的Rb激酶活性峰共同洗脱。对与cdk6共免疫沉淀的蛋白质分析表明，450 kDa复合物同时含有热休克蛋白90（Hsp90）和细胞分裂周期蛋白37（CDC37）。50 - 70 kDa复合物由两部分组成，一个66 kDa的复合物，包含与p19INK4d结合的cdk6和单体cdk6。通过制备细胞质和细胞核提取物分析了cdk6复合物的亚细胞定位。结果显示，450 kDa复合物主要存在于细胞质中，而170 kDa的细胞周期蛋白D/cdk6复合物和50 - 70 kDa复合物在细胞核和细胞质区室中均有存在。当对这些相同的提取物检测cdk6相关激酶活性时，只有细胞核中的cdk6复合物具有活性。这些数据表明，尽管在细胞质和细胞核中都可检测到cdk6/细胞周期蛋白D复合物，但只有细胞核中的cdk6具有活性。
