Identification of the protein kinases Pyk3 and Phg2 as regulators of the STATc-mediated response to hyperosmolarity

Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca(2+)-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3(-), phg2(-), and pyk3(-)/phg2(-) cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca(2+) liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3(-) and phg2(-) cells and even further decreased in pyk3(-)/phg2(-) cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2(-) cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues

Two <em>Dictyostelium</em> Tyrosine Kinase-Like kinases function in parallel, stress-induced STAT activation pathways

When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3

The dependence of dijet production on photon virtuality in ep collisions at HERA

The dependence of dijet production on the virtuality of the exchanged photon, Q^2, has been studied by measuring dijet cross sections in the range 0 < Q^2 < 2000 GeV^2 with the ZEUS detector at HERA using an integrated luminosity of 38.6 pb^-1. Dijet cross sections were measured for jets with transverse energy E_T^jet > 7.5 and 6.5 GeV and pseudorapidities in the photon-proton centre-of-mass frame in the range -3 < eta^jet <0. The variable xg^obs, a measure of the photon momentum entering the hard process, was used to enhance the sensitivity of the measurement to the photon structure. The Q^2 dependence of the ratio of low- to high-xg^obs events was measured. Next-to-leading-order QCD predictions were found to generally underestimate the low-xg^obs contribution relative to that at high xg^obs. Monte Carlo models based on leading-logarithmic parton-showers, using a partonic structure for the photon which falls smoothly with increasing Q^2, provide a qualitative description of the data.Comment: 35 pages, 6 eps figures, submitted to Eur.Phys.J.

Beauty photoproduction measured using decays into muons in dijet events in ep collisions at s\sqrt{s}=318 GeV

The photoproduction of beauty quarks in events with two jets and a muon has been measured with the ZEUS detector at HERA using an integrated luminosity of 110 pb$^{- 1}$. The fraction of jets containing b quarks was extracted from the transverse momentum distribution of the muon relative to the closest jet. Differential cross sections for beauty production as a function of the transverse momentum and pseudorapidity of the muon, of the associated jet and of $x_{\gamma}^{jets}$, the fraction of the photon's momentum participating in the hard process, are compared with MC models and QCD predictions made at next-to-leading order. The latter give a good description of the data.Comment: 32 pages, 6 tables, 7 figures Table 6 and Figure 7 revised September 200

Search for a narrow charmed baryonic state decaying to D^*+/- p^-/+ in ep collisions at HERA

A resonance search has been made in the D^*+/- p^-/+ invariant-mass spectrum with the ZEUS detector at HERA using an integrated luminosity of 126 pb^-1. The decay channels D^*+ -> D^0 pi^+_s -> (K^- pi^+) pi^+_s and D^*+ -> D^0 pi^+_s -> (K^- pi^+ pi^+ pi^-) pi^+_s (and the corresponding antiparticle decays) were used to identify D^*+/- mesons. No resonance structure was observed in the D^*+/- p^-/+ mass spectrum from more than 60000 reconstructed D^*+/- mesons. The results are not compatible with a report of the H1 Collaboration of a charmed pentaquark, Theta^0_c.Comment: 22 pages, 7 figures, 1 table; minor text revisions; 2 references adde

Wnt, Hedgehog and Junctional Armadillo/β-Catenin Establish Planar Polarity in the Drosophila Embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless (Wg or Wnt1), provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm

SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α

From Children to Adults: Motor Performance across the Life-Span

The life-span approach to development provides a theoretical framework to examine the general principles of life-long development. This study aims to investigate motor performance across the life span. It also aims to investigate if the correlations between motor tasks increase with aging. A cross-sectional design was used to describe the effects of aging on motor performance across age groups representing individuals from childhood to young adult to old age. Five different motor tasks were used to study changes in motor performance within 338 participants (7–79 yrs). Results showed that motor performance increases from childhood (7–9) to young adulthood (19–25) and decreases from young adulthood (19–25) to old age (66–80). These results are mirroring results from cognitive research. Correlation increased with increasing age between two fine motor tasks and two gross motor tasks. We suggest that the findings might be explained, in part, by the structural changes that have been reported to occur in the developing and aging brain and that the theory of Neural Darwinism can be used as a framework to explain why these changes occur