Paas Y, Fishelson Z
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Arch Biochem Biophys. 1995 Feb 1;316(2):780-8. doi: 10.1006/abbi.1995.1104.
Ecto-protein kinases (ecto-PK), primarily of the serine/threonine kinase type, have been previously described on the surface of various normal, transformed, and tumor cells. We have found that in the presence of ATP and Mg2+, exogenously added substrates such as phosvitin and poly(Glu4-Tyr) are phosphorylated by intact K562 erythroleukemia, HL60 promyelocytic leukemia, and U937 histiocytic leukemia human cells. Phosphoamino acid analysis indicated that phosvitin, histone H2B, casein, and protamine are phosphorylated on serine and threonine residues, whereas poly(Glu4-Tyr) is phosphorylated on tyrosine. We also present evidence showing that the C9 complement protein, a key component of the membranolytic protein complex of the complement system, is exclusively phosphorylated by the K562 cells on serine residues. Phosphorylation of poly(Glu4-Tyr) is markedly enhanced by Mn2+, whereas C9 phosphorylation is rather inhibited by Mn2+. It is concluded that human leukemic cells express on their surface two types of ecto-PK, one phosphorylating serines and threonines and one specific to tyrosines. The ecto-PKs are spontaneously shed from fully viable cells into the medium in a temperature-dependent manner. Upon sedimentation of cell supernatants at 100,000g, the ecto-PKs are found sedimented with small membrane vesicles. Treatment of intact K562 cells or of released membrane vesicles with bacterial phospholipase C, but not with trypsin or pronase, releases the two types of ecto-PK from the cell or vesicle membrane, respectively. This is accompanied by a marked increase in the released phosphorylating activity. It is, therefore, suggested that these ecto-PKs are either covalently linked to phospholipids or strongly attached to lipid-anchored molecules in the cell surface membrane. Several endogenous proteins in the released membranes are phosphorylated by the ecto-PKs on serines and to a lesser extend on threonines. Two proteins (PTP79 and PTP54) are phosphorylated in a manganese-dependent manner on tyrosines.
胞外蛋白激酶（ecto-PK）主要为丝氨酸/苏氨酸激酶类型，此前已在各种正常细胞、转化细胞和肿瘤细胞表面被描述。我们发现，在ATP和Mg2+存在的情况下，完整的K562红白血病细胞、HL60早幼粒细胞白血病细胞和U937组织细胞白血病人类细胞可使外源性添加的底物如卵黄高磷蛋白和聚（Glu4-Tyr）发生磷酸化。磷酸氨基酸分析表明，卵黄高磷蛋白、组蛋白H2B、酪蛋白和鱼精蛋白在丝氨酸和苏氨酸残基上发生磷酸化，而聚（Glu4-Tyr）在酪氨酸上发生磷酸化。我们还提供证据表明，补体系统溶膜蛋白复合物的关键成分C9补体蛋白仅在丝氨酸残基上被K562细胞磷酸化。聚（Glu4-Tyr）的磷酸化被Mn2+显著增强，而C9的磷酸化则被Mn2+相当程度地抑制。结论是人白血病细胞在其表面表达两种类型的胞外蛋白激酶，一种使丝氨酸和苏氨酸磷酸化，另一种对酪氨酸具有特异性。胞外蛋白激酶以温度依赖的方式从完全存活的细胞自发脱落到培养基中。在100,000g条件下对细胞上清液进行沉降时，发现胞外蛋白激酶与小膜泡一起沉降。用细菌磷脂酶C处理完整的K562细胞或释放的膜泡，但不用胰蛋白酶或链霉蛋白酶处理，可分别从细胞膜或膜泡膜上释放出两种类型的胞外蛋白激酶。这伴随着释放的磷酸化活性的显著增加。因此，提示这些胞外蛋白激酶要么与磷脂共价连接，要么与细胞表面膜中的脂锚定分子紧密结合。释放的膜中的几种内源性蛋白在丝氨酸上被胞外蛋白激酶磷酸化，在苏氨酸上的磷酸化程度较低。两种蛋白（PTP79和PTP54）在酪氨酸上以锰依赖的方式发生磷酸化。
