Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii

Abstract

The first step of the hexosamine pathway is the formation of glucosamine-6-phosphate from fructose-6-phosphate and L-glutamine, a reaction catalyzed by L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase) (14). In B. emersonii amidotransferase is a dimer of two apparently identical 76-kDa subunits. This enzyme exists in two forms, which are interconvertible by phosphorylation or dephosphorylation of the serine residue(s) (12,25). Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), the end product of hexosamine synthesis and also the substrate for chitin biosynthesis, specifically inhibits the activity of the phosphorylated form of the enzyme (12). In zoospores, the estimated concentration of UDP-GlcNAc (about 400 ,uM) is sufficient to completely inhibit the phosphorylated form of the amidotransferase (Ki for UDP-GlcNAc = 5 ,uM). Early during encystment, dephosphorylation of the enzyme allows it to escape inhibition by 25 (12,25). Although previous observations indicate that the dephosphorylation of amidotransferase is stimulated by magnesium (2, 12, 13), very little is known about the protein phosphatases (PPases) implicated in this process.The purpose of the present study was to characterize the PPases during the life cycle of B. emersondi and to identify those involved in the developmentally regulated control of hexosamine synthesis. To approach this problem we have used an improved procedure described by Cohen and coworkers for identifying and quantifying the serine-threonine PPase types 1, 2A, and 2C (PP1, PP2A, and PP2C) in animal tissues, yeast cells, plants, and protozoans (8, 9, 20, 22). Here we show that these three types of PPases are present in B. emersonii extracts and that amidotransferase is dephosphorylated by both PP2A and PP2C.

Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner

“…Thus the activity of GFAT is regulated by protein kinase(s) and phosphatase(s). In B. emersonii protein phosphatases 2A and 2C are involved in this process [9]. The stimulation of rat GFAT by protein kinase A (PKA) has been demonstrated in i o and in itro [10].…”

Global Gene Expression Analysis during Germination in the Chytridiomycete Blastocladiella emersonii

“…This phosphatase is also involved in many aspects of cellular function, including the regulation of metabolic enzymes and proteins involved in signal transduction (26). PP2A has been previously investigated for B. emersonii (4). The authors demonstrated that its activity is developmentally regulated, increasing during sporulation and reaching maximum levels in zoospores.…”

Searching for the role of protein phosphatases in eukaryotic microorganisms

“…Using a chitin-specific fluorescent dye, it was possible to demonstrate that okadaic acid does not inhibit cell wall synthesis (13). Previous work supported the idea that the regulation of cell wall synthesis was mainly at the level of amidotransferase, the first enzyme in the biosynthetic pathway responsible for the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), the substrate for chitin synthesis.…”