Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes
Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes
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DOI：
10.1074/jbc.m502464200
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发表时间：
2005-12-30
影响因子：
4.8
通讯作者：
Wadzinski, BE
中科院分区：
文献类型：
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作者：
Adams, DG;Coffee, RL;Wadzinski, BE
蛋白丝氨酸/苏氨酸磷酸酶2A（PP 2A）调节多种细胞信号转导途径。PP 2A在细胞中的主要形式是由支架（A）亚基、调节（B）亚基和催化（C）亚基组成的异源三聚体全酶。虽然已知PP 2A在该途径的多个步骤中调节Raf 1-MEK 1/2-ERK 1/2信号传导，但涉及的具体PP 2A全酶仍不清楚。为了解决这个问题，我们建立了四环素诱导的人胚肾293细胞系，与内源性水平相比，FLAG标记的B α/δ调节亚基的过表达接近3倍或B α的敲低超过70%。通过检测A和C亚基以及来自过表达FLAG-B α/δ亚基的细胞提取物的FLAG免疫复合物中的磷酸酶活性来证实功能性表位标记的B亚基的表达。使用MEK 1/2和ERK 1/2的磷酸化特异性抗体对细胞提取物进行Western分析表明，与对照细胞相比，这些激酶对表皮生长因子的应答激活在B α敲低细胞中显著降低，但在B α和B α过表达细胞中升高。与MEK 1/2和ERK 1/2的激活平行，Raf 1（Ser-259）的抑制性磷酸化位点在过表达B α或B α的细胞中被去磷酸化。药理学抑制剂研究以及转录因子Elk 1的ERK依赖性激活的报告基因测定表明，PP 2A全酶AB alpha C和AB delta C作用于Ras下游和MEK 1上游，以促进该MAPK信号级联的激活。此外，发现两种PP 2A全酶与Raf 1结合并催化抑制性磷酸-Ser-259的去磷酸化。总之，这些发现表明PP 2A AB α C和AB δ C全酶通过靶向Raf 1作为Raf 1-MEK 1/2-ERK 1/2信号传导的正调节剂起作用。
Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged B alpha/delta regulatory subunits by similar to 3-fold or knock-down of B alpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-B alpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in B alpha knock- down cells but elevated in B alpha- and B alpha- overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing B alpha or B alpha. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes AB alpha C and AB delta C act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A AB alpha C and AB delta C holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.