PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

BACKGROUND: Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. RESULTS: The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. CONCLUSION: Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered

Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-gamma treated cultures of Chlamydia pneumoniae

A common feature of many chlamydial infections is that they are often\ud asymptomatic and may persist for long periods of time if left untreated. In\ud addition to the well recognized lytic stage of the chlamydial developmental\ud cycle, evidence is now emerging to support a persistent phase in the cycle in\ud which the reticulate bodies are morphologically abnormal, viable but\ud non-infectious and presumably also have altered gene expression patterns. We\ud used an RT-PCR approach to study the differential levels of gene transcription\ud for 14 genes (16SrRNA, ompA, ompB, omcB, 76 kDa, gseA, pmp1, gltX, hsp60, yaeT,\ud pyk, nlpD, Cpn0585, Cpn1046) between normal and IFN-gamma treated Chlamydia\ud pneumoniae cell cultures. Even though the level of morphologically abnormal\ud reticulate bodies in our IFN-gamma treated cultures was low (approximately 10%\ud morphologically discernible, although presumably a larger percentage were in the\ud persistent state but not yet morphologically altered) we identified five genes\ud (ompA, ompB, pyk, nlpD, Cpn0585) that were clearly upregulated when compared to\ud normal cultures. This gene transcript profile may be characteristic of a general\ud stress state in Chlamydia, induced by IFN-gamma treatment in this case, but\ud perhaps more widely induced in other in vitro and in vivo situations

Paradoxical role of 3-methyladenine in pyocyanin-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells

The role of autophagy in pyocyanin (PCN)-induced toxicity in the central nervous system (CNS) remains unclear, with only evidence from our group identifying it as a mechanism underlying toxicity in 1321N1 astrocytoma cells. Therefore, the aim of this study was to further examine the role of autophagy in PCN-induced toxicity in the CNS. To achieve this, we exposed 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells to PCN (0-100 μmol/L) and tested the contribution of autophagy by measuring the impact of the autophagy inhibitor 3-methyladenine (3-MA) using a series of biochemical and molecular markers. Pretreatment of 1321N1 astrocytoma cells with 3-MA (5 mmol/L) decreased the PCN-induced acidic vesicular organelle and autophagosome formation as measured using acridine orange and green fluorescent protein-LC3 -LC3 fluorescence, respectively. Furthermore, 3-MA (5 mmol/L) significantly protected 1321N1 astrocytoma cells against PCN-induced toxicity. In contrast pretreatment with 3-MA (5 mmol/L) increased PCN-induced toxicity in SH-SY5Y neuroblastoma cells. Given the influence of autophagy in inflammatory responses, we investigated whether the observed effects in this study involved inflammatory mediators. The PCN (100 μmol/L) significantly increased the production of interleukin-8 (IL-8), prostaglandin E2 (PGE), and leukotriene B (LTB) in both cell lines. Consistent with its paradoxical role in modulating PCN-induced toxicity, 3-MA (5 mmol/L) significantly reduced the PCN-induced production of IL-8, PGE, and LTB in 1321N1 astrocytoma cells but augmented their production in SH-SY5Y neuroblastoma cells. In conclusion, we show here for the first time the paradoxical role of autophagy in mediating PCN-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells and provide novel evidence that these actions may be mediated by effects on IL-8, PGE, and LTB production

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen

A Novel Mammalian Retromer Component, Vps26B

The mammalian retromer protein complex, which consists of three proteins - Vps26, Vps29, and Vps35 - in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types

Infection of RAW264.7 cells by <i>B. pseudomallei</i> MSHR520, MSHR520Δ<i>bimA</i>, and MSHR520Δ<i>hcp</i>1.

<p>Infections were carried out over a period of 12 h and fixed for Giemsa staining (a) or lysed to release internalised bacteria, which were then plated for enumeration of cfu per well (b). (a) Formation of MNCs in cells infected with Δ<i>bimA</i> and Δ<i>hcp</i>1 mutants as compared with wt (MSHR520). Comparisons between mutants and wt were statistically significant (P<0.001). >1000 nuclei per well were counted and are expressed as number of nuclei in each category. Columns show the mean values with SEM. (b) Intracellular bacteria in cells infected with wt, Δ<i>bimA</i> and Δ<i>hcp</i>1 mutants as compared with wt (MSHR520). No significant difference (P = 0.46) is observed between wt and Δ<i>bimA</i> or wt and Δ<i>hcp</i>1. Scatter plots show each well as an individual point with the mean value represented by the horizontal bar. Data are derived from 4 separate experiments, each including duplicate or triplicate wells.</p

AHLs produced by <i>B. pseudomallei</i> MSHR520 and its derived mutants.

<p>Mutant strains are deleted in each AHL synthase gene indicated, either singly or in double or triple combination. Each value is presented as the mean ± SD. ND, not detected.</p

Representative views of <i>B. pseudomallei</i> infected RAW264.7 cells at 12 h post infection.

<p>A) & B) MSHR520 (wt) infected; C) MSHR520 Δ<i>bimA</i>; D) MSHR520 Δ<i>hcp</i>1; E) & F) MSHR520 Δ<i>bpsI</i>123. Nuclei are stained blue with DAPI; bacteria are labelled green with rabbit anti-<i>B. pseudomallei</i> IgG and anti-rabbit IgG-alexa 568; and actin is labelled red with phalloidin-alexa 488. Red actin tails, at the poles of bacteria, are visible in all panels with the exception of C. Scale bar represents 25 µm (A, C, D, E), 12.5 µm (B), 250 µm (F).</p

Infection of BALB/c mice with <i>B. pseudomallei</i> MSHR520 and related mutants.

<p>(a) Infection with MSHR520 and MSHR520Δ<i>bpsI</i>123 followed by determination of bacterial loads in systemic sites. (b) Infection with MSHR520Δcap and MSHR520Δcap Δ<i>bpsI</i>123 followed by determination bacterial loads in the upper respiratory tract (URT), olfactory epithelium (OE), olfactory bulb (OB), nasal-associated lymphoid tissue (NALT), and blood. No significant differences were observed between wt and mutant strains for equivalent tissues and time points.</p