Searching for the role of protein phosphatases in eukaryotic microorganisms.

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism

Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa.

A cAMP-activatable Ca2+-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

Serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from Neurospora crassa.

The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type-1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PP1 activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor-1 (IC50 2 nM). On the other hand. PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-1. About 80% of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.

Serine/threonine protein phosphatases and a protein phosphatase 1 inhibitor from Neurospora crassa

The major spontaneously active serine/threonine (Ser/Thr) protein phosphatase activities in N. crassa wild type (FGSC 424) were type 1 (PP1), type-2A (PP2A) and type-2C (PP2C). PP1 and PP2C predominantly dephosphorylated phosphorylase a and casein, respectively. PP2A acted on both substrates, but was two-fold more active against casein. PPI activity was inhibited by protamine, heparin, okadaic acid (IC50 50 nM) and mammalian inhibitor- 1 (lC50 2 nM). On the other hand, PP2A activity was inhibited by much lower concentrations of okadaic acid (IC50 0.2 nM) and also by protamine, but not by heparin or inhibitor-l. About 80 per cent of total PP1 activity was associated with the particulate fraction and could be partially extracted with 0.5 M NaCl. Seventy and ninety percent of PP2A and PP2C activities, respectively, were found in the soluble fraction. In addition we have partially purified an acid and thermostable PP1 inhibitor which effectively inhibits both N. crassa and mammalian PP1.

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

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.