Bodenbach L, Fauss J, Robitzki A, Krehan A, Lorenz P, Lozeman F J, Pyerin W
German Cancer Research Center, Heidelberg.
Eur J Biochem. 1994 Feb 15;220(1):263-73. doi: 10.1111/j.1432-1033.1994.tb18622.x.
Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in the regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta); second, the autophosphorylation of the holoenzyme at the N-terminus of CKII beta, suspected to be involved in tuning of the kinase activity. Whether CKII alpha and CKII alpha' reconstitute comparably with CKII beta to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKII beta were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKII beta stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant alpha 2 beta 2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKII beta and CKII alpha in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKII beta autophosphorylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKII beta mutants was comparable both to each other and to that of wild-type CKII beta. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2-5-fold with no major differences between CKII alpha and CKII alpha' stimulation. Calmodulin phosphorylation by both CKII alpha and CKII alpha' was decreased similarly by CKII beta and the CKII beta mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKII beta or CKII beta mutants. The data suggest that CKII alpha and CKII alpha' form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autophosphorylation status of CKII beta.
人酪蛋白激酶II（CKII）是一种广泛存在且具有多种功能的丝氨酸/苏氨酸激酶，参与细胞生长和分化的调控。从生化角度来看，有两个特征尤为显著：其一，由两个催化亚基（α和/或α'）和两个调节亚基（β）组成的四聚体结构；其二，全酶在CKII β的N端发生自身磷酸化，推测这与激酶活性的调节有关。目前尚不清楚CKII α和CKII α'与CKII β重新组装形成全酶的情况是否相似。为了进行系统研究，在大肠杆菌中表达了全套重组CKII亚基以及CKII β的自身磷酸化突变体，并进行了比较重组实验。以1:1的摩尔比，CKII β以卵黄高磷蛋白作为底物时，对两个催化亚基的刺激作用大致增强了五倍。两种重组的CKII同工型所达到的活性水平与从人胎盘中分离得到的全酶处于同一数量级。这也与通过含有串联排列的CKII β和CKII α编码区的双顺反子构建体在大肠杆菌中表达得到的重组α2β2全酶相似。Ser2和Ser3均被确定为自身磷酸化位点；通过寡核苷酸介导的定点诱变将其中之一替换为丙氨酸仅影响CKII β自身磷酸化的程度，两者都替换则导致自身磷酸化丧失。尽管存在这些差异，但所有CKII β突变体的刺激作用彼此相当，且与野生型CKII β的刺激作用相当。当使用除卵黄高磷蛋白以外的其他底物，如微管蛋白或上游结合因子（UBF）时，也得到了相同的结果。然而，刺激程度具有底物特异性，在CKII α和CKII α'的刺激作用之间，范围为2至5倍，没有明显差异。CKII β和CKII β突变体同样降低了CKII α和CKII α'对钙调蛋白的磷酸化作用。诸如环磷酸腺苷反应元件结合蛋白（CREB）、人乳头瘤病毒16 E7或Jun等蛋白质，在单独存在时均不会被任何一个催化亚基磷酸化，但在CKII β或CKII β突变体存在时，它们都会成为两者的底物。这些数据表明，CKII α和CKII α'形成了相似的CKII全酶，并且全酶活性的调节与CKII β的自身磷酸化状态无关。
