The main objective of this project is to characterize protein kinases previously identified as regulators of glycogen metabolism in Neurospora crassa. In addition, we are interested in the identification of protein quinases that phosphorylates the enzyme glycogen synthase (GSN). In a previous work we used a set of mutant strains in genes encoding protein kinases to identify protein quinases likely involved in glycogen metabolism regulation. Many proteins were identified, some of them were already functionally characterized in different organisms and most of them without biochemical and functional characterization. Some of the mutant strains showed changes in GSN phosphorylation based on the results of enzyme activity and the presence of different isoforms when analyzed by 2D-PAGE followed by Western blot. This work aims to deeply investigate the subject by performing the following studies: 1- to investigate which protein kinases from the crude cellular extract are able to phosphorylate "in vitro" GSN by MudPIT analysis, 2- to investigate the role of the NcPho85-like protein, a cyclin-dependent protein kinase, and its putative cyclin partner in the GSN phosphorylation, 3- to investigate which proteins are target for phosphorylation by the NCU06419 e NCU02234 ORFs produts using a phosphoproteome approach. Both proteins belong to the MAP kinase protein family and were previously identified in the screening. The results will be important to bring new information on how phosphorylation events regulate glycogen metabolism, either acting in different targets that results in regulation or directly phosphorylating glycogen synthase, the more phosphorylated regulatory enzyme.
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