Wahl M I, Jones G A, Nishibe S, Rhee S G, Carpenter G
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
J Biol Chem. 1992 May 25;267(15):10447-56.
We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.
我们之前已证明磷脂酶C-γ1（PLC-γ1）催化活性的酪氨酸磷酸化依赖性调节（西贝，S.，瓦尔，M. I.，埃尔南德斯-索托马约尔，S. M. T.，汤克斯，N. K.，李，S. G.，和卡彭特，G.（1990年）《科学》250卷，1253 - 1256页）。A - 431细胞中PLC-γ1催化活性的增加迅速发生，在表皮生长因子（EGF）刺激后5分钟达到最大激活。某些其他生长因子（成纤维细胞生长因子、血小板衍生生长因子）也刺激PLC-γ1催化活性，而胰岛素则不然。在来自EGF处理细胞的可溶性（胞质溶胶）和颗粒性（膜）制剂中均观察到PLC-γ1比活性有类似的增加（2 - 3倍）。在来自EGF处理的A - 431细胞的裂解物的胞质溶胶和膜部分中均检测到酪氨酸磷酸化的PLC-γ1，但酪氨酸磷酸化的PLC-γ1在胞质溶胶中的比例（约50%）高于膜中的比例（约20%）。因为在该测定中，胶束浓度的非离子去污剂Triton X - 100可检测到PLC-γ1催化活性的酪氨酸磷酸化依赖性增加，所以我们使用以各种摩尔比溶解在Triton X - 100中的底物磷脂酰肌醇4,5 - 二磷酸（PtdIns 4,5 - P2）评估了从对照或EGF处理细胞的胞质溶胶中免疫沉淀的PLC-γ1的动力学性质。对照酶与EGF激活的酶在Ks和Km方面的行为有所不同。对照酶的Ks值比激活酶高7.5倍（分别为1.5 mM和0.22 mM）。EGF激活也是PLC-γ1催化的PtdIns 4,5 - P2水解的正别构调节剂，即激活的酶表现出明显的米氏动力学，Km为0.6摩尔分数的PtdIns 4,5 - P2，而对照酶在PtdIns 4,5 - P2水解方面表现出S形动力学。在低底物摩尔分数（例如0.07）时，对照酶的反应速度比激活酶低4倍。然而，在高底物摩尔分数（例如0.33）时，两种形式的PLC-γ1的估计最大反应速度（Vmax）相当。通过增加纳摩尔浓度的Ca2+，来自对照和EGF处理细胞的PLC-γ1活性均受到刺激。尽管在每个测试的Ca2+浓度下，来自EGF处理细胞中PLC-γ1的催化活性都大于对照PLC-γ1，但在Ca2+浓度高于约300 nM时，活性的相对刺激明显更大。
