Meggio F, Agostinis P, Pinna L A
Biochim Biophys Acta. 1985 Aug 30;846(2):248-56. doi: 10.1016/0167-4889(85)90072-2.
We have shown by gel filtration on Sepharose 4B at low ionic strength that casein kinases S (type 1), heparin-insensitive, and TS (type 2), heparin-inhibited, of rat liver cytosol participate in two distinct multimolecular systems, Ve/Vo = 1.25 and Ve/Vo = 1.90, respectively, both less retarded than the peak of cAMP-dependent protein kinase activity (Ve/Vo = 2.04). Both casein kinase I and casein kinase II complexes are unstable in 0.5 M NaCl, giving rise by gel filtration under these conditions to the free forms of casein kinase S (Ve/Vo = 2.37, Mr 34 000) and casein kinase TS (Ve/Vo = 2.10, Mr 130 000), respectively. In contrast, the elution volume of cAMP-dependent protein kinase activity is always the same irrespective of the ionic strength of the medium. Casein kinase I, accounting for the whole casein kinase S activity of cytosol, also contains a phosphorylatable 31-kDa protein (p31) which is a substrate of casein kinase S, since its phosphorylation is insensitive to heparin, the heat-stable inhibitor and trifluoperazine, but it is prevented by beryllium. Casein kinase II, on the other hand, apparently results from the association of the whole casein kinase TS (type 2) of rat liver cytosol with a 90-kDa protein substrate (p90) which is distinct from glycogen synthase according to their different peptide mappings. The radiolabelling of p90 is inhibited by heparin, unlabeled GTP and polyglutamates, while it is dramatically and specifically enhanced by polylysine. At least three more protein bands of Mr 58 000, 52 000 and 37 000 are phosphorylated by casein kinase TS in the casein kinase II fraction: their co-elution with casein kinase TS, however, seems to be accidental and their radiolabeling in the presence of polylysine is almost negligible compared to that of p90. It is concluded that p31 and p90 may represent specific targets of casein kinase S and casein kinase TS, respectively, whose intimate association with the enzymes could be functionally significant.
我们已经通过在低离子强度下于琼脂糖4B上进行凝胶过滤实验表明，大鼠肝细胞溶胶中的酪蛋白激酶S（1型，对肝素不敏感）和酪蛋白激酶TS（2型，受肝素抑制）分别参与两个不同的多分子系统，洗脱体积与总体积之比（Ve/Vo）分别为1.25和1.90，两者的滞留程度均小于环磷酸腺苷（cAMP）依赖性蛋白激酶活性的峰值（Ve/Vo = 2.04）。酪蛋白激酶I和酪蛋白激酶II复合物在0.5M氯化钠中均不稳定，在这些条件下通过凝胶过滤分别产生酪蛋白激酶S的游离形式（Ve/Vo = 2.37，相对分子质量34000）和酪蛋白激酶TS的游离形式（Ve/Vo = 2.10，相对分子质量130000）。相比之下，无论介质的离子强度如何，cAMP依赖性蛋白激酶活性的洗脱体积始终相同。占肝细胞溶胶中酪蛋白激酶S全部活性的酪蛋白激酶I还含有一种可磷酸化的31kDa蛋白（p31），它是酪蛋白激酶S的底物，因为其磷酸化对肝素、热稳定抑制剂和三氟拉嗪不敏感，但铍可抑制其磷酸化。另一方面，酪蛋白激酶II显然是由大鼠肝细胞溶胶中整个酪蛋白激酶TS（2型）与一种90kDa蛋白底物（p90）结合形成的，根据它们不同的肽图谱，该底物与糖原合酶不同。p90的放射性标记受到肝素、未标记的鸟苷三磷酸（GTP）和聚谷氨酸的抑制，而聚赖氨酸则能显著且特异性地增强其放射性标记。在酪蛋白激酶II组分中，至少还有另外三条相对分子质量为58000、52000和37000的蛋白带被酪蛋白激酶TS磷酸化：然而，它们与酪蛋白激酶TS的共洗脱似乎是偶然的，并且与p90相比，如果存在聚赖氨酸，它们的放射性标记几乎可以忽略不计。得出的结论是，p31和p90可能分别代表酪蛋白激酶S和酪蛋白激酶TS的特异性靶点，它们与这些酶的紧密结合可能在功能上具有重要意义。
