Tolwinski N S, Shapiro P S, Goueli S, Ahn N G
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
J Biol Chem. 1999 Mar 5;274(10):6168-74. doi: 10.1074/jbc.274.10.6168.
Stimulation of mammalian cells results in subcellular relocalization of Ras pathway enzymes, in which extracellular signal-regulated protein kinases rapidly translocate to nuclei. In this study, we define conditions for nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) by examining effects of perturbing the nuclear export signal (NES), the regulatory phosphorylation sites Ser218 and Ser222, and a regulatory domain at the N terminus. After disrupting the NES (Delta32-37), nuclear uptake of MKK was enhanced when quiescent cells were activated with serum-phorbol 12-myristate 13-acetate or BXB-Raf-1 cotransfection. Uptake was enhanced by mutation of Ser218 and Ser222 to Glu and Asp, respectively, and blocked by mutation of these residues to Ala, although mutation of Lys97 to Met, which renders MKK catalytically inactive, did not interfere with uptake. Therefore, nuclear uptake of MKK requires incorporation of phosphate or negatively charged residues at the activation lip but not enzyme activity. On the other hand, uptake of an active MKK mutant with disrupted NES (Delta32-51) was elevated in quiescent as well as stimulated cells, and pretreatment of cells with the MKK inhibitor 1,4-diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio]butadiene blocked nuclear uptake. Thus, signaling downstream of MKK is also necessary for translocation. Finally, wild type MKK containing an intact NES translocates to nuclei during mitosis before envelope breakdown. Comparison of mutants with Ser to Glu and Asp or Ala substitutions indicates that Ser phosphorylation is also required for mitotic nuclear uptake of MKK.
对哺乳动物细胞的刺激会导致Ras信号通路酶在亚细胞水平重新定位，其中细胞外信号调节蛋白激酶会迅速转移至细胞核。在本研究中，我们通过检测干扰核输出信号（NES）、调节性磷酸化位点Ser218和Ser222以及N端调节结构域的影响，来确定丝裂原活化蛋白激酶激酶1（MKK1）核定位的条件。破坏NES（Δ32 - 37）后，当静止细胞用血清 - 佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯或BXB - Raf - 1共转染激活时，MKK的核摄取增强。分别将Ser218和Ser222突变为Glu和Asp可增强摄取，而将这些残基突变为Ala则会阻断摄取，尽管将Lys97突变为Met使MKK催化失活，但并不影响摄取。因此，MKK核摄取需要在激活环处掺入磷酸基团或带负电荷的残基，但不需要酶活性。另一方面，NES破坏的活性MKK突变体（Δ32 - 51）在静止细胞和受刺激细胞中的摄取均升高，并且用MKK抑制剂1,4 - 二氨基 - 2,3 - 二氰基 - 1,4 - 双[2 - 氨基苯硫基]丁二烯预处理细胞可阻断核摄取。因此，MKK下游信号传导对于转运也是必需的。最后，含有完整NES的野生型MKK在有丝分裂期间核膜破裂前转移至细胞核。将Ser突变为Glu、Asp或Ala的突变体比较表明，MKK有丝分裂核摄取也需要Ser磷酸化。
