Song W X, Vacca M F, Welti R, Rintoul D A
Department of Biochemistry, Kansas State University, Manhattan 66506-4901.
J Biol Chem. 1991 Jun 5;266(16):10174-81.
Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
此前有报道（布雷默，E.G.，施莱辛格，J.，以及羽田森，S.-I.（1986年）《生物化学杂志》261卷，2434 - 2440页）称，神经节苷脂GM3在经Triton X - 100处理的人表皮样癌（A431）细胞膜制剂中，可抑制表皮生长因子（EGF）刺激的EGF受体磷酸化。此外，这些作者还报道GM3可抑制A431细胞的生长。相反，一种修饰的神经节苷脂，去N - 乙酰GM3，可增强EGF受体的EGF依赖性酪氨酸激酶活性。在本研究及后续研究中（花井，N.，土肥，T.，诺雷斯，G.A.，以及羽田森，S.-I.（1988年）《生物化学杂志》263卷，6296 - 6301页），在存在Triton X - 100的情况下测定了A431细胞膜受体的酪氨酸激酶活性。在本报告中，我们证实，在存在Triton X - 100的情况下，GM3抑制而去N - 乙酰GM3刺激EGF受体自身磷酸化。然而，在不存在去污剂的情况下，神经节苷脂GM3抑制EGF刺激的受体自身磷酸化，而去N - 乙酰GM3对EGF刺激的受体自身磷酸化没有影响。在A431细胞质膜以及通过冻融法对[32P]ATP通透的3T3细胞膜中，在用阿拉霉素通透的完整A431细胞中，以及在存在[32P]正磷酸盐的情况下生长的完整A431细胞中，均测定了这些神经节苷脂对受体自身磷酸化的影响。因此，GM3对受体自身磷酸化的抑制作用在存在和不存在去污剂的情况下均得到证实；而去N - 乙酰GM3的刺激作用仅在存在去污剂的情况下观察到。我们还证明，神经节苷脂GM3可抑制表达人EGF受体基因的转染鼠成纤维细胞（3T3）的EGF刺激生长（韦卢，T.J.，贝吉诺特，L.，瓦斯，W.C.，张，K.，帕斯坦，I.，以及洛维，D.R.（1989年）《细胞生物化学杂志》39卷，153 - 166页）。去N - 乙酰神经节苷脂GM3对这些细胞的生长没有影响。缺乏内源性EGF受体的对照成纤维细胞（普鲁斯，R.M.，以及赫施曼，H.R.（1977年）《美国国家科学院院刊》74卷，3918 - 3921页）的生长不受任何一种神经节苷脂存在的影响。同样，神经节苷脂GM3而非去N - 乙酰神经节苷脂GM3可抑制转染成纤维细胞的EGF依赖性[3H]胸腺嘧啶核苷掺入DNA。标记的胸腺嘧啶核苷掺入对照成纤维细胞的DNA不受任何一种神经节苷脂存在的影响。这些研究表明，神经节苷脂GM3而非其脱乙酰类似物可在完整膜中影响EGF受体激酶活性。（摘要截短至400字）
