The C subunit of Dictyostelium CAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI.Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequencecomparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe-motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

The cAMP-dependent protein kinase (cAPK) plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum. The presence of an open reading frame on the gene, pkaC (previously named either Dd PK2 or Dd PK3 by different groups), predicts a 73-kDa polypeptide with 54% similarity to the catalytic subunits of cAPKs from other organisms. Using anti-peptide antibodies, we show that the pkaC gene product, PkaC, is a 73-kDa polypeptide. Despite the fact that PkaC is about twice the size of its mammalian counterparts, it possesses all of the properties required of a catalytic subunit. It is physically associated with the regulatory subunit, and this association results in an inhibition of the catalytic activity which is reverted by cAMP. PkaC copurifies with cAPK activity, and an increased cAPK activity is observed in cells overexpressing PkaC. We conclude that PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK and discuss the unusual features of this protein with the highest molecular weight of known cAPKs.

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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.

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Delta mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V(max) of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Delta mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Delta mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade.

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.