Candido Thiago De Souza, Gonçalves Rodrigo Duarte, Felício Ana Paula, Freitas Fernanda Zanolli, Cupertino Fernanda Barbosa, De Carvalho Ana Carolina Gomes Vieira, Bertolini Maria Célia
*Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista (UNESP), 14800-900, Araraquara, SP, Brazil.
Biochem J. 2014 Dec 15;464(3):323-34. doi: 10.1042/BJ20140942.
Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.
糖原在多种生物体中作为碳水化合物储备发挥作用，其代谢受到高度调控。糖原合成酶和糖原磷酸化酶分别是合成和降解过程的限速酶，它们的活性通过别构调节和可逆磷酸化来调控。为了鉴定影响粗糙脉孢菌糖原代谢的蛋白激酶，我们对84个丝氨酸/苏氨酸激酶敲除菌株进行了筛选。我们鉴定出了多种已被描述为在不同生物体中控制糖原代谢的激酶，如NcSNF1、NcPHO85、NcGSK3、NcPKA、PSK2同源物和NcATG1。此外，许多假定的激酶也与糖原代谢的控制有关。还鉴定出了两种激酶NcIME - 2和NcNIMA，它们已被进行功能表征，但与糖原代谢调控无关。在鉴定出的激酶中，值得一提的是NcSNF1的作用。我们在本研究中表明，该激酶与糖原合成酶的磷酸化有关，与野生型菌株相比，Ncsnf1KO菌株在生长过程中积累的糖原水平更高，糖原合成酶（GSN）±葡萄糖6 - 磷酸活性比值更高，且磷酸化的GSN同工型较少，这证明了这一点。结果使我们得出结论，在粗糙脉孢菌中，这种激酶直接或间接促进糖原合成酶的磷酸化，这与酿酒酵母中所描述的情况相反。这些激酶在基因表达调控中也发挥作用，因为编码去分支酶的基因gdn被筛选中鉴定出的蛋白质下调。一些激酶影响生长和发育，这表明糖原代谢与细胞生长和发育之间存在联系。
