Ragione F D, Russo G L, Oliva A, Mercurio C, Mastropietro S, Pietra V D, Zappia V
Institute of Biochemistry of Macromolecules, Medical School, Second University of Naples, Italy.
J Biol Chem. 1996 Jul 5;271(27):15942-9. doi: 10.1074/jbc.271.27.15942.
The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition.
D型细胞周期蛋白依赖性激酶（CDK4和CDK6）活性的调节似乎是真核细胞通过G1细胞周期阶段进程中的关键步骤。参与这一过程的机制之一是一些分子量在14至20 kDa之间的小蛋白抑制剂与这些CDK结合。我们评估了正常细胞和转化细胞中两种此类抑制剂，即p16(INK4)和p18的含量，以及包含这些蛋白质的大分子复合物的生化特性。所得结果表明：（i）与p16(INK4)基因不同，p18基因的表达不受pRb状态的调节；（ii）p16(INK4)和p18基因的表达之间不存在明显关系；（iii）这两种蛋白质的大量分子并未与CDK结合，而是以游离分子形式存在；（iv）每种抑制剂与CDK蛋白以1:1的化学计量比形成复合物；（v）细胞周期蛋白D和抑制剂蛋白之间存在对CDK蛋白的竞争，导致无法检测到细胞内游离激酶。此外，利用人天然部分纯化的p16(INK4)或纯重组蛋白，我们能够在体外证明CDK4 - 细胞周期蛋白D1复合物的解离以及CDK4 - p16(INK4)双分子复合物的形成。我们的研究结果表明，在细胞分裂周期中，p16(INK4)蛋白家族成员和细胞周期蛋白D竞争与CDK4/CDK6结合，并且它们的定量比例对于G1期向S期的转变至关重要。
