Biochemical characterization of recombinant Drosophila type 1 serine/threonine protein phosphatase (PP1c) produced in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express Drosophila melanogaster type 1beta serine/threonine phosphoprotein phosphatase catalytic subunit (PP1beta9C). A construct encoding PP1beta9C with a short NH(2)-terminal fusion including six histidine residues was introduced into the X-33 and KM71H strains of P. pastoris by homologous recombination. Recombinant protein was purified from cell free extracts 24 h after methanol induction. PP1beta9C was purified to a specific activity of 12,077 mU/mg by a three-step purification method comprising (NH(4))(2)SO(4)-ethanol precipitation followed by Ni(2+)-agarose affinity chromatography and Mono Q anion-exchange chromatography. This purification scheme yielded approximately 80 microg of active, soluble PP1beta9C per 1 L of culture. In contrast to recombinant PP1beta9C overexpressed in bacteria, which differs from native PP1c in several biochemical criteria including the requirement for divalent cations, sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity, recombinant PP1beta9C produced in P. pastoris has native-like properties. P. pastoris thus provides a reliable and convenient system for the production of active, native-like recombinant PP1beta9C.

Protein phosphatase 1beta is required for the maintenance of muscle attachments.

Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle [1] [2] [3] [4] [5]. Drosophila and humans both have multiple genes encoding PP1 isoforms [3] [6] [7]; each has one beta and several alpha isoform genes (alpha(1), alpha(2), alpha(3) in flies, alpha and gamma in humans; mammalian PP1beta is also known as PP1delta). The alpha/beta subtype differences are highly conserved between flies and mammals [6]. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, we show here that the Drosophila beta isoform has a distinct biological role. We show that PP1beta9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs) [8]. We have isolated a new, semi-lethal flw allele that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate.

The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue.

Phosphatase inhibitor-2 (I-2) is a mammalian phosphoprotein that binds to the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1c) and inhibits its activity in vitro. Recombinant PP1c differs from native PP1c in several biochemical criteria, including the requirement for Mn(2+), sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity. I-2 can convert recombinant PP1c into a native-like activity in vitro. It has therefore been suggested that I-2 may act as a molecular chaperone for PP1 in vivo. We have identified a Drosophila homologue (I-2Dm) in a two-hybrid screen for PP1c-binding proteins. The sequence of I-2Dm is 35% identical with that of I-2, whereas the catalytic subunits themselves are >85% identical in flies and humans; however, we show that many biochemical properties of I-2 are conserved. Like I-2, I-2Dm can convert recombinant PP1c to a native-like activity. This strongly suggests that this ability is an essential, conserved role of I-2 and I-2Dm.

Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa.

Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PPI and PP2A, respectively). inhibit Neurospora crassa hyphal growth. When N. crassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34 degrees C than at 25 degrees C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The first, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa.

pzl-1 encodes a novel protein phosphatase-Z-like Ser/Thr protein phosphatase in Neurospora crassa.

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa.

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.

Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa.

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster.

The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.

Comparative characterization of liver glycogen metabolism in rat and guinea-pig.

1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca(2+)-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-1 is phosphorylated by cyclic AMP-dependent protein kinase.