Matsuoka M, Kato J Y, Fisher R P, Morgan D O, Sherr C J
Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Mol Cell Biol. 1994 Nov;14(11):7265-75. doi: 10.1128/mcb.14.11.7265-7275.1994.
The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
由调节性D型细胞周期蛋白和细胞周期蛋白依赖性激酶（cdk）组成的功能性全酶的组装，是哺乳动物体细胞周期G1期进程的限速步骤。D型细胞周期蛋白与其主要催化亚基cdk4之间的复合物在催化上是无活性的，直到与细胞周期蛋白结合的cdk4在单个苏氨酰残基（Thr-172）上发生磷酸化。这一步骤由一种细胞周期蛋白依赖性激酶激活激酶（CAK）催化，该酶在功能上类似于在Thr-161/160位点磷酸化cdc2和cdk2的酶。在这里，我们证明小鼠cdc2/cdk2 CAK的催化亚基（一种命名为p39MO15的39 kDa蛋白）可以与昆虫或哺乳动物细胞中存在的一种调节蛋白组装，以产生一种能够磷酸化并酶促激活与各自细胞周期蛋白伴侣结合的cdk2和cdk4的CAK活性。一种新鉴定的37 kDa细胞周期蛋白样蛋白（细胞周期蛋白H [R. P. Fisher和D. O. Morgan，《细胞》78:713 - 724，1994]）可以与p39MO15组装，在体外激活细胞周期蛋白A-cdk2和细胞周期蛋白D-cdk4，这意味着CAK在结构上让人联想到细胞周期蛋白-cdk复合物本身。针对p39MO15亚基产生的抗血清可以完全耗尽哺乳动物细胞裂解物中细胞周期蛋白A-cdk2和细胞周期蛋白D-cdk4的CAK活性，而在所得免疫复合物中活性得以恢复。通过使用免疫复合物CAK测定法，在静止细胞中以及在整个细胞周期中均恒定地检测到细胞周期蛋白A-cdk2和细胞周期蛋白D-cdk4的CAK活性。因此，尽管CAK对于细胞周期蛋白-cdk复合物的酶促激活至关重要，但它似乎既不是细胞从静止状态出现的限速因素也不受刺激性有丝分裂原的上游调节控制。
