Waskiewicz A J, Johnson J C, Penn B, Mahalingam M, Kimball S R, Cooper J A
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Mol Cell Biol. 1999 Mar;19(3):1871-80. doi: 10.1128/MCB.19.3.1871.
Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.
真核生物翻译起始因子4E（eIF4E）与mRNA的5'帽结合，并使mRNA与其他蛋白质合成起始因子及核糖体形成复合物。哺乳动物eIF4E的活性对于带帽mRNA的翻译很重要，并且被认为受两种机制调控。首先，在静止细胞中，eIF4E被诸如4EBP1等结合蛋白隔离。有丝分裂原通过刺激4EBP1的磷酸化诱导eIF4E的释放。其次，有丝分裂原和应激诱导eIF4E在Ser 209位点磷酸化，增加eIF4E对带帽mRNA及相关支架蛋白eIF4G的亲和力。我们之前表明，一种有丝分裂原和应激激活的激酶Mnk1，在体外可在生理位点磷酸化eIF4E。在此我们表明，Mnk1在体内调控eIF4E的磷酸化。Mnk1直接与eIF4G结合，并与eIF4G和eIF4E共纯化。我们鉴定出Mnk1中的激活磷酸化位点，并构建了显性负性和激活突变体。显性负性Mnk1的表达降低有丝分裂原诱导的eIF4E磷酸化，而激活的Mnk1的表达增加基础eIF4E磷酸化。激活的突变体Mnk1还在过表达4EBP1的细胞中诱导eIF4E广泛磷酸化。这表明eIF4E的磷酸化是由Mnk1或细胞中一种非常相似的激酶催化的，并且独立于从4EBP1释放eIF4E的其他有丝分裂信号。
