Interaction with protein phosphatase 1 Is essential for bifocal function during the morphogenesis of the Drosophila compound eye.

The gene bifocal (bif), required for photoreceptor morphogenesis in the Drosophila compound eye, encodes a protein that is shown to interact with protein phosphatase 1 (PP1) using the yeast two-hybrid system. Complex formation between Bif and PP1 is supported by coprecipitation of the two proteins. Residues 992 to 995 (RVQF) in the carboxy-terminal region of Bif, which conform to the consensus PP1-binding motif, are shown to be essential for the interaction of Bif with PP1. The interaction of PP1 with bacterially expressed and endogenous Bif can be disrupted by a synthetic peptide known to block interaction of other regulatory subunits with PP1. Null bif mutants exhibit a rough eye phenotype, disorganized rhabdomeres (light-gathering rhodopsin-rich microvillar membrane structures in the photoreceptor cells) and alterations in the actin cytoskeleton. Expression of wild-type bif transgenes resulted in significant rescue of these abnormalities. In contrast, expression of transgenes encoding the Bif F995A mutant, which disrupts binding to PP1, was unable to rescue any aspect of the bif phenotype. The results indicate that the PP1-Bif interaction is critical for the rescue and that a major function of Bif is to target PP1c to a specific subcellular location. The role of the PP1-Bif complex in modulating the organization of the actin cytoskeleton underlying the rhabdomeres is discussed.

The Drosophila melanogaster homologue of the human BBC1 gene is highly expressed during embryogenesis.

We have previously reported the isolation and preliminary characterisation of a full-length cDNA sequence derived from the human BBC1 gene, a gene which displays differential expression in tumours of the female breast [Adams et al., Hum. Mol. Genet. 1 (1992) 91-96]. Here, we report the isolation and characterisation of the Drosophila melanogaster (Dm) homologue of this human gene. The Dmbbc1 cDNA is 62% identical to the human BBC1 cDNA within a conserved open reading frame and the encoded proteins share 74% sequence similarity. The Dmbbc1 mRNA is expressed at all stages of Dm development, with the highest levels of expression occurring during embryogenesis. In addition, the Dm and human BBC1 proteins share remarkable degrees of identity with the products of recently isolated plant and avian bbc1 cDNAs. The sequences of all the predicted BBC1 proteins are highly similar to that of the rat ribosomal subunit protein L13 [Olvera and Wool, Biochem. Biophys. Res. Commun. 201 (1994) 102-107], strongly indicating that the BBC1 protein is ribosomal protein L13.

Drosophila melanogaster protein phosphatase inhibitor-2: identification of a site important for PP1 inhibition.

A database search with human protein phosphatase inhibitor-2 (I-2) identified a Drosophila melanogaster cDNA that encoded a protein identical in length and sharing 39% amino acid identity (58% similarity) with human I-2. The mRNA encoding this protein is expressed in both sexes and throughout development, unlike Drosophila inhibitor-t. The bacterially expressed protein was a specific inhibitor of protein phosphatase 1 with an IC(50) of <1 nM, confirming that it is the Drosophila homologue of mammalian inhibitor-2. Mutation of Phe residues conserved in I-2 from lower and higher eukaryotes showed that Phe-33 is important for inhibition of PP1c.

Characterisation of a novel Drosophila melanogaster testis specific PP1 inhibitor related to mammalian inhibitor-2: identification of the site of interaction with PP1.

A novel Drosophila melanogaster protein, termed inhibitor-t, that bears 41% sequence similarity to human protein phosphatase inhibitor-2 has been identified using human protein phosphatase 1 (PP1) in the yeast two hybrid system. Inhibitor-t mRNA is detected in adult males, larvae and pupae and the 184 amino acid thermostable protein located only in testis. The gene for inhibitor-t maps to cytological location 86F1 on the third chromosome. Bacterially expressed inhibitor-t specifically inhibits both mammalian and D. melanogaster PP1 catalytic subunits with an IC50 of approximately 200 nM. A motif -FEX1X2RK-, conserved between inhibitor-t, inhibitor-2 and its Saccharomyces cerevisiae homologue Glc8, is demonstrated to be required for binding to PP1.

Cloning of the complete coding region for human protein phosphatase inhibitor 2 using the two hybrid system and expression of inhibitor 2 in E. coli.

The yeast two hybrid system has been employed to identify cDNAs encoding proteins which interact with the gamma 1 isoform of human protein phosphatase 1. Here we report the isolation of cDNA encoding human protein phosphatase inhibitor. The deduced human sequence of 205 amino acids shows 92% identity to inhibitor 2 from rabbit. Human inhibitor 2 was expressed in E. coli and purified to homogeneity. The expressed human protein inhibited both native and bacterially expressed PP1, with the same Ki (1 nM) as inhibitor 2 purified from skeletal muscle. A gene or pseudogene for inhibitor 2 may be present near the major histocompatibility complex on chromosome 6.

Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53.

The p53 binding protein, termed p53BP2, was identified as a protein interacting with protein phosphatase 1 (PP1) in the yeast two hybrid system. The interaction was confirmed by co-immunoprecipitation of p53BP2 with epitope-tagged PP1 in vitro. The p53BP2-PP1 complex was stable to NaCl at concentrations which dissociate the p53-p53BP2 complex, and the binding of PP1 and p53 to p53BP2 was mutually exclusive. The region required for interaction with PP1 was shown to be contained within amino acids 297-431 of p53BP2, which includes two ankyrin repeats. The phosphorylase phosphatase activity of PP1 was inhibited by p53BP2 at nanomolar concentrations. These results suggest that PP1 may be involved in dephosphorylation and regulation of p53 through interaction with p53BP2.

5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC.

Human protein phosphatase-2C alpha (PP2C alpha) was purified to homogeneity after expression in Escherichia coli. AMP inhibited the dephosphorylation of AMP-activated protein kinase (AMPK), but not phosphocasein, by PP2C alpha. The concentration dependence and the effects of other nucleotides (ATP and formycin A-5'-monophosphate) suggest that AMP acts by binding to the same site which causes direct allosteric activation of AMPK. A similar, although less pronounced, effect was observed with another protein phosphatase (PP2AC). We have now shown that AMPK activates the AMPK cascade by four mechanisms, which should make the system exquisitely sensitive to changes in AMP concentration.

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.

NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1.

The cell cycle-regulated protein serine/threonine NIMA-related kinase 2 (Nek2), which shows a predominant localization at centrosomes, is identified as a protein which interacts with protein phosphatase 1 (PP1) using the yeast two-hybrid system. Complex formation between Nek2 and PP1 is supported by co-precipitation of the two proteins using transfected expression constructs of Nek2 and the endogenous Nek2/PP1 proteins. The sequence KVHF in the C-terminal region of Nek2, which conforms to the consensus PP1-binding motif, is shown to be essential for the interaction of Nek2 with PP1. Nek2 activity increases with autophosphorylation and addition of phosphatase inhibitors and decreases in the presence of PP1. PP1 is a substrate for Nek2 and phosphorylation of PP1gamma(1) on two C-terminal sites reduces its phosphatase activity. The presence of a ternary complex containing centrosomal Nek2-associated protein (C-Nap1), Nek2 and PP1 has also been demonstrated, and C-Nap1 is shown to be a substrate for both Nek2 and PP1 in vitro and in cell extracts. The implications of kinase-phosphatase complex formation involving Nek2 and PP1 are discussed in terms of the coordination of centrosome separation with cell cycle progression.

Isolation and characterization of a novel gene with differential expression in benign and malignant human breast tumours.

We report the identification of a novel cDNA representing an mRNA showing significantly higher levels of expression in benign breast lesions than in carcinomas. This cDNA was identified by differential screening of a cDNA library generated from a breast carcinoma, and shows consistently higher expression in fibroadenomas than in carcinomas. The expression in both benign and malignant tissues is highest in epithelial cells as determined by in situ hybridization to tissue sections. The nucleotide sequence of the full-length cDNA has been determined, and the deduced protein is highly basic with no signal or transmembrane sequence, but two potential nuclear localization signals. Neither the DNA nor the protein sequence show any significant homology to sequences in current databases. The cDNA hybridizes to multiple sequences within both human and other mammalian genomes, but to single genomic sequences in Drosophila, Physarum and Schizosaccharomyces pombe. This cDNA therefore represents a highly conserved gene sequence. We have identified only one major transcript in human cells, and it seems likely that there are several pseudogenes within the human genome.

Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos.

The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, approximately 25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.

Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit.

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.