The divine horseman in the art of the western Roman Empire

This thesis examines the image of the mounted god in the western provinces of the Roman Empire. Artists in the provinces often turned to Roman, actually Graeco-Roman, models when developing an iconographical form for local deities. The thesis investigates the transformation of Graeco-Roman imagery and looks at the way artists adapted the Roman forms which would have become familiar to them with the advance of the Roman army and Roman settlement in the provinces of the western part of the empire. The mounted gods discussed are the Celtic Mars and Jupiter, whose Roman names conceal their Celtic identity, Epona, the Dioscuri in the western Empire, the Thracian Rider and the Danubian Rider cult

The enzymes of the glyoxylate bypass operon of Escherichia coli ML308

1. The glyoxylate bypass allows Escherichia coli to generate precursors for biosynthesis during growth on acetate. During operation of the bypass there is competition for isocitrate between isocitrate dehydrogenase (ICDH) and isocitrate lyase (ICL). Previous studies had shown that ICDH is regulated by a phosphorylation/dephosphorylation mechanism. However, ICL and its possible contribution to regulation at this branchpoint had not been studied. 2. A quick, reliable procedure was developed for purification of ICL from acetate-grown Escherichia coli ML308. 10-12 mg of pure enzyme could be obtained from 20g wet weight of bacteria. The specific activity was 35-40mumol/min/mg of protein at 37 C. 3. The of the native enzyme was 177 000, determined by gel filtration and 180 000, determined by analytical ultracentrifugation. The subunit Mr. was 45 000. This implies that Escherichia coli ICL is a tetramer. 4. The optimum pH for ICL activity was pH 7.3, within the physiological range for Escherichia coli. 5. The kinetics of binding of substrates, products and/or their analogues was studied for the cleavage and the condensation reactions of ICL. The results show that, unlike the enzyme from other sources, ICL from Escherichia coli obeys a random order mechanism in which an enzyme-isocitrate-succinate ternary complex can be formed. 6. The Km of ICL for Ds -isocitrate was 0.063mM at pH7. 3. The Km was sensitive to changes in pH and also to the presence of inorganic anions such as Cl- and SO2-. 7. Several compounds were found to inhibit ICL but these effects could be ascribed to structural similarities between the inhibitors and the substrates for the enzyme. 2-oxoglutarate and phosphoenolpyruvate were shown to be succinate analogues and 3-phosphoglycerate was shown to be a glyoxylate analogue. The intracellular concentration of 3-phosphoglycerate suggests that this compound may inhibit ICL significantly in intact cells. However, none of the effects observed could be attributed to the existence of an allosteric regulatory site. 8. There was no evidence that ICL is a phosphorylated molecule nor that ICDH kinase/phosphatase can affect ICL. 9. A recombinant plasmid carrying an 11 kilobase Clal-Clal fragment of genomic DNA which complements an aceA (ICL) mutation was constructed by Dr. E. M. T. El-Mansi. Measurement of enzyme activities in crude cell extracts and in vitro transcription-translation experiments showed that this plasmid (pEM9) encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (MS-A), ICL and ICDH kinase/phosphatase and in that order. 10 ICL was purified from the overexpressing strain KAT-1/pEM9 following the procedure developed for ICL from Escherichia coli ML308 up to the phenyl-Sepharose step. ICL from both sources was identical by peptide-mapping, Km, NH2-terminal sequence and amino acid composition. The pI of both ICLs was 4. 4 as determined by chromatofocusing on a Fast Protein Liquid Chromatography Mono P column. 11. Overexpression of MS-A in Escherichia coli KAT-1/pEM9 meant that it could be distinguished from MS-B and a purification was developed for MS-A from Escherichia coli KAT-l/pEM9. The N-terminal amino acid sequence of MS-A was determined. 12. ICDH kinase/phosphatase from Escherichia coli KAT-1/pEM9 was subjected to limited proteolysis by trypsin. Proteolysis proceeded in two stages in which the first cleavage product had lost its ICDH phosphatase activity but retained its ICDH kinase activity. The second cleavage product was completely devoid of both kinase and phosphatase activities. These results could mean that the active sites for ICDH kinase and ICDH phosphatase are different

Identification of 2R-ohnologue gene families displaying the same mutation-load skew in multiple cancers

The complexity of signalling pathways was boosted at the origin of the vertebrates, when two rounds of whole genome duplication (2R-WGD) occurred. Those genes and proteins that have survived from the 2R-WGD—termed 2R-ohnologues—belong to families of two to four members, and are enriched in signalling components relevant to cancer. Here, we find that while only approximately 30% of human transcript-coding genes are 2R-ohnologues, they carry 42–60% of the gene mutations in 30 different cancer types. Across a subset of cancer datasets, including melanoma, breast, lung adenocarcinoma, liver and medulloblastoma, we identified 673 2R-ohnologue families in which one gene carries mutations at multiple positions, while sister genes in the same family are relatively mutation free. Strikingly, in 315 of the 322 2R-ohnologue families displaying such a skew in multiple cancers, the same gene carries the heaviest mutation load in each cancer, and usually the second-ranked gene is also the same in each cancer. Our findings inspire the hypothesis that in certain cancers, heterogeneous combinations of genetic changes impair parts of the 2R-WGD signalling networks and force information flow through a limited set of oncogenic pathways in which specific non-mutated 2R-ohnologues serve as effectors. The non-mutated 2R-ohnologues are therefore potential therapeutic targets. These include proteins linked to growth factor signalling, neurotransmission and ion channels

Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573

Fasting and systemic insulin signaling regulate phosphorylation of brain proteins that modulate cell morphology and link to neurological disorders

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease

Differential roles and regulation of the protein kinases PAK4, PAK5 and PAK6 in melanoma cells

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell–cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAF(V600E) or NRAS(Q61K) mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC–PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer

Disruption of the AMPK-TBC1D1 nexus increases lipogenic gene expression and causes obesity in mice via promoting IGF1 secretion

Tre-2/USP6, BUB2, cdc16 domain family member 1 (the TBC domain is the GTPase activating protein domain) (TBC1D1) is a Rab GTPase activating protein that is phosphorylated on Ser(231) by the AMP-activated protein kinase (AMPK) in response to intracellular energy stress. However, the in vivo role and importance of this phosphorylation event remains unknown. To address this question, we generated a mouse model harboring a TBC1D1(Ser231Ala) knockin (KI) mutation and found that the KI mice developed obesity on a normal chow diet. Mechanistically, TBC1D1 is located on insulin-like growth factor 1 (IGF1) storage vesicles, and the KI mutation increases endocrinal and paracrinal/autocrinal IGF1 secretion in an Rab8a-dependent manner. Hypersecretion of IGF1 causes increased expression of lipogenic genes via activating the protein kinase B (PKB; also known as Akt)–mammalian target of rapamycin (mTOR) pathway in adipose tissues, which contributes to the development of obesity, diabetes, and hepatic steatosis as the KI mice age. Collectively, these findings demonstrate that the AMPK–TBC1D1 signaling nexus interacts with the PKB–mTOR pathway via IGF1 secretion, which consequently controls expression of lipogenic genes in the adipose tissue. These findings also have implications for drug discovery to combat obesity