Marianne Gamper

Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor

T.Araki and M.Gamper contributed equally to this work

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild‐type cells. Time‐lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature‐sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium.

Mechanistic Insights into the Isochorismate Pyruvate Lyase Activity of the Catalytically Promiscuous PchB from Combinatorial Mutagenesis and Selection

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM‐deficient E. coli strain, but only if produced without the predicted N‐terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis–Menten kinetics, having a kcat of 50 s−1 and a Km of 180 µm (at 30 °C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size‐exclusion chromatography. Secondary‐structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all‐α‐helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram‐positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.

The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein

The arginine deiminase (ADI) pathway in Pseudomonas aeruginosa serves to generate ATP. The three enzymes involved, ADI, catabolic ornithine carbamoyltransferase and carbamate kinase, are induced by oxygen limitation and encoded by the contiguous arcABC genes. A 1.5-kb region upstream from arcABC was sequenced and found to contain an open reading frame, arcD, coding for a hydrophobic polypeptide of 52 kDa. The content and distribution of hydrophobic amino acids suggest that the arcD gene product may be a transmembrane protein. When arcD was fused to an Escherichia coli promoter, the ArcD protein was synthesized in E. coli maxicells and detected in the membrane fraction. In sodium dodecyl sulfate-polyacrylamide-gel electrophoresis the ArcD protein migrated like a 32-kDa protein; such anomalous electrophoretic mobility is known for other highly hydrophobic proteins. Mutations in arcD rendered the cells unable to utilize extracellular arginine as an energy source. Since anaerobic arginine consumption and ornithine release are coupled in P. aeruginosa, it is proposed that arcD specifies an arginine: ornithine antiporter or a part thereof. Insertions of IS21 or Tn1725 in arcD had a strong polar effect on the expression of the arcAB enzymes, indicating that the arc genes are organized as an arcDABC operon.

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQδ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.

Multiple roles of the novel protein tyrosine phosphatase PTP3 during Dictyostelium growth and development.

PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic protein tyrosine phosphatase domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow-growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys to Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of various sizes. Antiphosphotyrosine Western blot (immunoblot) analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with nonnutrient buffer led to reciprocal changes in the pattern of several phosphotyrosine proteins, including a protein of approximately 130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested that p130 is a potential substrate of PTP3. A transient posttranslational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data obtained strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2.

Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development.

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase.

STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer Differentiation inducing factor 1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 overexpressing cells. Conversely, overexpression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induces a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation, whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation.

Regulation of Dictyostelium Protein-tyrosine Phosphatase-3 (PTP3) through Osmotic Shock and Stress Stimulation and Identification of pp130 as a PTP3 Substrate

Osmotic shock and growth-medium stimulation ofDictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the ∼130-kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein-tyrosine phosphatase-3 (PTP3). This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine-phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment.