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Mapping the functional domains of elongation factor-2 kinase

机译：绘制延伸因子2激酶的功能域

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A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., ct al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci, U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that art: not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R35]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation, eEF-2 was the only known substrate for eEF-3 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-I. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The aminoterminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-3, targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36". [References: 36]
机译：最近鉴定了与丝氨酸/苏氨酸/酪氨酸蛋白激酶超家族成员不同源的一类新的真核蛋白激酶[Futey，L.M。，等。 （1995）生物化学杂志。化学270，523-529; Ryazanov，A. G.等。 （1997年）过程。 Natl。学院Sci，U.S.A。94，4884-4889]。该类包括真核伸长因子2激酶，双歧杆菌球蛋白肌球蛋白重链激酶A，B和C，以及一些尚未被充分鉴定的哺乳动物推定的蛋白激酶[Ryazanov，A. G.，等。 （1999）Curr。生物学9，R43-R35]。 eEF-2激酶是一种普遍存在的蛋白激酶，可磷酸化并灭活真核翻译延伸因子-2，因此可以调节翻译过程中多肽链延伸的速率，eEF-2是eEF-3激酶的唯一已知底物。我们在这里证明，eEF-2激酶可以有效地磷酸化Dictyostelium肌球蛋白重链中的肌球蛋白重链激酶A磷酸化位点的16个氨基酸的肽MH-1。这使我们能够开发出一种快速测定eEF-2激酶活性的方法。为了定位eEF-2激酶的功能域，我们在人类大肠杆菌中以GST标记的融合蛋白表达了人类eEF-2激酶，然后进行了系统的体外缺失诱变。我们分析了eEF-2激酶缺失突变体的自磷酸化能力，以及将eEF-2和肽底物MH-1磷酸化的能力。在氨基酸51和3​​35之间缺失的突变体不能自磷酸化，也不能磷酸化eEF-2和MH-1。在氨基酸521和725之间缺失的突变体不能磷酸化eEF-2，但是仍然能够自磷酸化和磷酸化MH-1。氨基酸2和50之间以及336和520之间缺失的激酶能够催化所有三个反应。此外，在共沉淀测定中，单独表达的C末端结构域（氨基酸336-725）与eEF-2结合。这些结果表明，eEF-2激酶由通过接头区域连接的两个结构域组成。氨基末端结构域包含催化结构域，而羧基末端结构域包含eEF-3靶向结构域。钙调蛋白结合区位于氨基酸51和96之间。eEF-2激酶的羧基末端结构域的氨基酸序列与几种蛋白质显示相似性，所有这些蛋白质均包含我们命名的36个氨基酸基序的重复序列“主题36”。 [参考：36]

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