Kornbluth S, Sebastian B, Hunter T, Newport J
Department of Biology, University of California, San Diego, La Jolla 92093.
Mol Biol Cell. 1994 Mar;5(3):273-82. doi: 10.1091/mbc.5.3.273.
The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.
进入有丝分裂的关键调节因子是丝氨酸/苏氨酸激酶p34cdc2。这种激酶通过与细胞周期蛋白结合以及在多个位点的磷酸化来调节。酪氨酸15和苏氨酸14位点的磷酸化被认为会抑制cdc2的激酶活性。在粟酒裂殖酵母中，wee1（可能还有mik1）蛋白激酶催化酪氨酸15位点的磷酸化。目前尚不清楚是这些蛋白激酶还是其他尚未确定的蛋白激酶使苏氨酸14磷酸化。在本报告中，我们利用非洲爪蟾卵提取物表明，催化苏氨酸14位点磷酸化的激酶与膜紧密结合。具体而言，我们已表明，在没有细胞质的情况下，纯化的膜组分可促进cdc2在苏氨酸14和酪氨酸15位点的磷酸化。相比之下，细胞质只能使cdc2在酪氨酸15位点磷酸化，这表明至少存在两种定位明显不同的催化cdc2酪氨酸15位点磷酸化的激酶亚群。膜相关的酪氨酸15和苏氨酸14激酶活性在盐提取或去污剂提取过程中的表现相似，在细胞周期以及在DNA复制时延迟有丝分裂的检查点机制作用下的调节方式也相似。这表明存在一种双特异性膜相关蛋白激酶可能催化酪氨酸15和苏氨酸14位点磷酸化的可能性。
