Romero-Oliva F, Allende J E
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile.
J Cell Biochem. 2001;81(3):445-52. doi: 10.1002/1097-4644(20010601)81:3<445::aid-jcb1058>3.0.co;2-2.
Protein kinase CK2 is a ubiquitous protein that phosphorylates multiple substrates and is composed of catalytic (alpha, alpha') and regulatory (beta) subunits. Abundant evidence relates CK2 to the regulation of cell division. p21(WAF1/CIP1) is a potent inhibitor of cyclin-dependent kinases and of DNA replication and acts as a key inhibitor of cell cycle progression. In this work we examine the relation between these two important proteins. The interaction between the CK2 beta regulatory subunit of CK2 and p21(WAF1/CIP1) has been confirmed. Using a pull-down assay and fusion constructs of glutathione transferase with fragments of CK2 beta and other mutants, it was possible to define that the N-terminal (1-44) portion of CK2 beta contains a p21(WAF1/CIP1) binding site. CK2 reconstituted from recombinant alpha and beta subunits can phosphorylate p21(WAF1/CIP1) in vitro. This phosphorylation is greatly enhanced by histone H1. p21(WAF1/CIP1) can inhibit the phosphorylation of substrate casein by CK2. This inhibition, however, seems to be due to competition by p21(WAF1/CIP1) as an alternate substrate since in order to observe inhibition it is necessary that the concentration of p21 be of the same order of magnitude as the casein substrate concentration. This competition is not related to the binding of p21(WAF1/CIP1) to CK2 beta because it can also be observed when, in the absence of CK beta, CK alpha is used to phosphorylate casein in the presence of the p21.
蛋白激酶CK2是一种普遍存在的蛋白质，可磷酸化多种底物，由催化亚基（α、α'）和调节亚基（β）组成。大量证据表明CK2与细胞分裂的调控有关。p21（WAF1/CIP1）是细胞周期蛋白依赖性激酶以及DNA复制的强效抑制剂，是细胞周期进程的关键抑制剂。在这项研究中，我们研究了这两种重要蛋白质之间的关系。已证实CK2的β调节亚基与p21（WAF1/CIP1）之间存在相互作用。使用下拉实验以及谷胱甘肽S-转移酶与CK2β片段和其他突变体的融合构建体，能够确定CK2β的N端（1-44）部分含有一个p21（WAF1/CIP1）结合位点。由重组α亚基和β亚基重构的CK2在体外可磷酸化p21（WAF1/CIP1）。组蛋白H1可大大增强这种磷酸化作用。p21（WAF1/CIP1）可抑制CK2对底物酪蛋白的磷酸化作用。然而，这种抑制作用似乎是由于p21（WAF1/CIP1）作为替代底物的竞争所致，因为为了观察到抑制作用，p21的浓度必须与酪蛋白底物浓度处于同一数量级。这种竞争与p21（WAF1/CIP1）与CK2β的结合无关，因为在没有CKβ的情况下，当使用CKα在p21存在下磷酸化酪蛋白时也可观察到这种竞争。
