Kraft A S, Anderson W B
J Biol Chem. 1983 Aug 10;258(15):9178-83.
We have addressed the question of the possible presence of calcium-activated phospholipid-dependent protein kinase (Ca2+-PL protein kinase) activity in undifferentiated embryonal carcinoma cells, and if this activity might be altered during differentiation to a parietal endoderm cell type. Undifferentiated nullipotent F9 embryonal carcinoma cells, as well as differentiated parietal endoderm cells (PYS-2), were utilized. Using an in vitro assay with histone H1 as phosphate acceptor, Ca2+-PL protein kinase activity could not be found in the 100,000 X g supernatant prepared from either cell type. However, passage of 100,000 X g supernatant from PYS cells over a DEAE-cellulose column revealed Ca2+-PL protein kinase activity which eluted with 0.045 M NaCl. The partially purified PYS enzyme has an approximate Mr = 70,000 as determined by Sephadex G-150 gel filtration, and exhibits an apparent Ka for Ca2+ of 32 microM. The PYS Ca2+-PL protein kinase also exhibits a requirement for Mg2+, with maximal activity noted at 10 mM Mg2+. This enzyme is stimulated by acidic phospholipids, while neutral phospholipids such as phosphatidylcholine have little effect. Diacylglycerol markedly increased histone H1 phosphorylation in the presence of Ca2+ and phospholipid. Unlike that of PYS cells, when the 100,000 X g supernatant prepared from F9 cells was passed over a DEAE-cellulose column no Ca2+-PL protein kinase activity could be found in the eluted fractions. Previously it has been reported that exposure of F9 cells to all-trans-retinoic acid induces differentiation to a parietal endoderm cell type. Treatment of F9 cells with 0.1 microM retinoic acid provoked a time-dependent increase in cytosolic Ca2+-PL protein kinase activity as measured after DEAE-cellulose chromatography of the 100,000 X g supernatant. This increase in Ca2+-PL protein kinase activity correlates with differentiation to the parietal endoderm cell type. These findings indicate that cytosolic Ca2+-PL protein kinase activity is very low, or nonexistent, in undifferentiated embryonal carcinoma stem cells. With differentiation to a parietal endoderm cell type there is a marked increase in soluble Ca2+-PL protein kinase activity which exhibits properties similar to those described for this enzyme in other differentiated tissues.
我们研究了未分化的胚胎癌细胞中是否可能存在钙激活磷脂依赖性蛋白激酶（Ca2+-PL蛋白激酶）活性，以及在分化为壁层内胚层细胞类型的过程中这种活性是否会发生改变。我们使用了未分化的全能F9胚胎癌细胞以及分化的壁层内胚层细胞（PYS-2）。以组蛋白H1作为磷酸受体进行体外测定，在这两种细胞类型制备的100,000×g上清液中均未发现Ca2+-PL蛋白激酶活性。然而，将PYS细胞的100,000×g上清液通过DEAE-纤维素柱后，发现有Ca2+-PL蛋白激酶活性，其在0.045 M NaCl中洗脱。通过Sephadex G-150凝胶过滤测定，部分纯化的PYS酶的近似分子量Mr = 70,000，其对Ca2+的表观解离常数Ka为32 microM。PYS Ca2+-PL蛋白激酶对Mg2+也有需求，在10 mM Mg2+时活性最高。该酶受酸性磷脂刺激，而中性磷脂如磷脂酰胆碱几乎没有影响。在有Ca2+和磷脂存在的情况下，二酰基甘油显著增加组蛋白H1的磷酸化。与PYS细胞不同，当将F9细胞制备的100,000×g上清液通过DEAE-纤维素柱时，在洗脱组分中未发现Ca2+-PL蛋白激酶活性。此前有报道称，将F9细胞暴露于全反式维甲酸会诱导其分化为壁层内胚层细胞类型。用0.1 microM维甲酸处理F9细胞，在对100,000×g上清液进行DEAE-纤维素柱层析后测定，胞质Ca2+-PL蛋白激酶活性随时间依赖性增加。Ca2+-PL蛋白激酶活性的这种增加与向壁层内胚层细胞类型的分化相关。这些发现表明，在未分化的胚胎癌干细胞中，胞质Ca2+-PL蛋白激酶活性非常低或不存在。随着向壁层内胚层细胞类型的分化，可溶性Ca2+-PL蛋白激酶活性显著增加，其性质与在其他分化组织中描述的该酶的性质相似。
