Properties and regulation of a transiently assembled ERK2•Ets-1 signaling complex
Properties and regulation of a transiently assembled ERK2•Ets-1 signaling complex
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DOI：
10.1021/bi0610451
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发表时间：
2006-11-21
期刊：
影响因子：
2.9
通讯作者：
Dalby, Kevin N.
中科院分区：
文献类型：
--
作者：
Callaway, Kari A.;Rainey, Mark A.;Dalby, Kevin N.
ERK 2是一种脯氨酸导向的蛋白激酶，其对底物Ets-1上的单个苏氨酸（Thr-38）显示出高度特异性，该苏氨酸位于共有序列（36）phi-chi-Thr-Pro（39）内（其中phi通常是小的疏水残基，chi似乎不受限制）。Thr-38位于一个长的柔性N-末端尾（残基1-52），其中还含有第二个潜在的磷酸化位点，Ser-26。Ets-1如何结合ERK 2以促进Thr-38的磷酸化，同时区别Ser-26的磷酸化尚不清楚。为了描述ERK 2对Ets-1的分子识别的细节，通过荧光各向异性分析了Ets-1的各种突变体和截短体的结合。所获得的数据支持这样的观点，即N-末端尾部包含一个以前未识别的对接位点，该位点促进Thr-38的磷酸化。这个新的对接位点有助于组装Ets-1和ERK 2的复合物，并对复合物的稳定性做出类似的贡献，就像Ets-1的尖结构域一样。MKK 1在体外激活ERK 2诱导了激活片段（DFG-APE）的大的构象转变，但既不诱导ERK 2的自缔合，也不破坏ERK 2，Ets-1复合物的稳定性。后一种观察结果表明，活性位点固有的相互作用对于复合物组装并不重要，这一观点进一步得到以下观察结果的支持：许多不同氨基酸取代Pro-39不会使复合物不稳定。环13内ERK 2的突变表明Ets-1结合底物结合沟。这些数据表明，ERK 2使用两个弱对接相互作用来特异性地组装复合物，也许这样做会阻止Ser-26进入活性位点。Ets-1的残基1-138（Ets Delta 138）被来自Elk-1的肽N-QKGKPRDLELPLSPSL-C从ERK 2置换，表明Ets-1接合ERK 2的D-募集位点（β 7-β 8反向转角和α D-α E螺旋）。Ets Delta 138被衍生自Elk-1的肽N-AKLSFQFPS-C从ERK 2置换，表明Ets Delta 138也与ERK 2的F-募集位点通讯。
ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine ( Thr-38) on the substrate Ets-1, which lies within the consensus sequence (36)phi-chi-Thr-Pro(39) ( where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail ( residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment ( DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2, Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 ( Ets Delta 138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta 7-beta 8 reverse turn and the alpha D-alpha E helix) of ERK2. Displacement of Ets Delta 138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that Ets Delta 138 communicates with the F-recruitment site of ERK2 also.