Randez-Gil F, Sanz P, Entian K D, Prieto J A
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt am Main, Germany.
Mol Cell Biol. 1998 May;18(5):2940-8. doi: 10.1128/MCB.18.5.2940.
The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PH is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with lambda-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2 (S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.
HXK2基因对于酿酒酵母中导致发酵代谢适应的多种调节作用是必需的。然而，Hxk2p在这些作用中的具体分子基础仍不清楚。为了理解己糖激酶PH的生理功能，一个重要特征是它是一种磷蛋白，因为蛋白质磷酸化在真核细胞的大多数代谢信号转导中至关重要。在此我们表明，Hxk2p在体内以二聚体 - 单体平衡存在，该平衡受磷酸化影响。只有单体形式被磷酸化，而二聚体则没有。Hxk2p的可逆磷酸化依赖于碳源，在如半乳糖、棉子糖和乙醇等劣质碳源上更为广泛。添加葡萄糖后，Hxk2p在体内的去磷酸化作用增强。在葡萄糖阻遏突变体cat80/grr1、hex2/reg1和cid1/glc7中不存在这种效应。用λ - 磷酸酶处理来自cid1 - 226（glc7 - T152K）突变细胞的葡萄糖粗提物，会显著降低磷蛋白的存在，这表明CID1/GLC7磷酸酶与其调节性HEX2/REG1亚基一起参与了Hxk2p单体的去磷酸化。编码第15位丝氨酸变为丙氨酸的HXK2突变[HXK2 (S15A)]是为了阐明己糖激酶PII磷酸化的体内功能。在这个突变体中，Hxk2蛋白无法进行磷酸化，细胞无法实现对蔗糖酶的葡萄糖阻遏。表达突变酶的细胞中HXT基因表达的葡萄糖诱导也受到影响。尽管我们不能排除细胞代谢状态缺陷作为这些现象的起源，但我们的结果表明己糖激酶的磷酸化在体内对于葡萄糖信号转导至关重要。
