Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15.
Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15.
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DOI：
10.1021/bi200202y
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发表时间：
2011-05-31
期刊：
影响因子：
2.9
通讯作者：
Dalby KN
中科院分区：
文献类型：
--
作者：
Kaoud TS;Devkota AK;Harris R;Rana MS;Abramczyk O;Warthaka M;Lee S;Girvin ME;Riggs AF;Dalby KN
细胞外信号调节蛋白激酶，ERK 2，完全激活的磷酸化和没有His 6标签，显示几乎没有倾向于二聚化或不含钙或镁离子时，通过光散射或分析ultracentravation分析。光散射显示，约90%的ERK 2是单体。含或不含镁（10 mM）的沉降平衡数据（在4.8-11.2 μM ERK 2下获得）通过理想单组分模型进行了良好描述，拟合摩尔质量为40，180 ± 240 Da（-Mg 2+离子）和41，290 ± 330 Da（+Mg 2+离子）。这些值接近于41，711 Da的序列衍生质量，表明在溶液中没有发生显著的ERK 2二聚化。使用增强的货车Holde-Weischet方法分析15 μM ERK 2溶液的沉降速度数据，确定在含或不含10 mM MgCl 2的情况下，活化ERK 2的沉降系数约为3.22 S。根据沉降速度和平衡数据计算的摩擦系数比（f/f0）为1.28，接近于约42 kDa的球状蛋白的预期值。由实验测定的摩尔质量和沉降系数计算的平移扩散系数约为8.3 × 10-7 cm ~ 2s ~（-1），与动态光散射法测定的钙或镁离子存在和不存在时的值以及NMR光谱法测定的值一致。已经提出ERK 2同源二聚化并且仅结合细胞质而非核蛋白。然而，我们的光散射数据显示，ERK 2与细胞质支架蛋白PEA-15形成约57 kDa的强1：1复合物。因此，ERK 2作为单体结合PEA-15。我们的数据提供了有力的证据，ERK 2是单体在生理条件下。对具有非生理性His 6-Tag的相同ERK 2构建体的分析显示在相同离子条件下基本上二聚化。
The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His6-tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~ 90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8–11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40,180 ± 240 Da (- Mg2+ ions) and 41,290 ± 330 Da (+ Mg2+ ions). These values, close to the sequence-derived mass of 41,711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~ 3.22 S for activated ERK2 with or without 10 mM MgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for a globular protein of ~ 42 kDa. The translational diffusion coefficient of ~ 8.3 × 10-7 cm2s-1 calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~ 57 kDa with the cytoplasmic scaffold protein PEA-15. Thus ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the non-physiological His6-Tag shows substantial dimerization under the same ionic conditions.
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DOI：
10.1016/j.jchromb.2006.07.031
发表时间：
2006-12-05
影响因子：
3
作者：
Amor-Mahjoub, Mouna;Suppini, Jean-Philippe;Ladjimi, Moncef
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影响因子：
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影响因子：
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