Plotnikov Alexander, Chuderland Dana, Karamansha Yael, Livnah Oded, Seger Rony
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel.
Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel.
Cell Physiol Biochem. 2019;53(2):366-387. doi: 10.33594/000000144.
BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear.
To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK.
Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation.
These results provide an important role of CK2 in regulating nuclear ERK activities.
背景/目的：细胞外信号调节激酶（ERK）1和2是丝裂原活化蛋白激酶（MAPK）家族的成员。受到刺激后，这些激酶从细胞质转移至细胞核，在细胞核中诱导增殖和分化等生理过程。这种激酶的转移机制涉及核转运信号（NTS）内两个丝氨酸残基的磷酸化，这使得其能够与输入蛋白7结合并随后通过核孔进入细胞核。然而，这一过程的调控机制以及所涉及的蛋白激酶尚不清楚。
为了解决这一问题，我们研发了特异性抗磷酸化-SPS抗体，在蛋白质印迹中使用该抗体及其他抗体，并使磷酸化模拟突变型ERK结晶。
我们在此表明，两个丝氨酸残基的磷酸化主要由酪蛋白激酶2（CK2）介导，并且活性ERK可能有助于N端丝氨酸的磷酸化。我们还证明，磷酸化依赖于ERK从细胞质锚定蛋白上的释放。磷酸化模拟型ERK的晶体结构显示，NTS磷酸化在ERK中产生了一个酸性区域。我们的模型是，在静息细胞中，ERK与细胞质锚定蛋白结合，这会阻止其NTS磷酸化。受到刺激后，ERK TEY结构域的磷酸化会释放ERK，并允许CK2和活性ERK对其NTS进行磷酸化，从而在ERK中产生一个带负电荷的区域，使其与输入蛋白7结合并发生核转运。
这些结果揭示了CK2在调节细胞核内ERK活性方面的重要作用。
