Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2

Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.     

Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes.     

Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares

The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 microg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from -0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4+/-0.1 cm in control group, 1.5+/-0.1 in oxytocin and carbetocin group and 1.3+/-0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Intermediate filaments in muscle and epithelial cells of nematodes

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.     

Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli

A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic ¹³C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h⁻¹. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO₂. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h⁻¹ was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.