Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.     

Leukotactin-1-induced ERK activation is mediated via Gi/Go protein/PLC/PKC delta/Ras cascades in HOS cells

Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.     

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.     

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72

A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample. This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7. The maximum activity was observed at pH 8.7 and 50 °C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain. The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

Molecular network and functional implications of macromolecular tRNA synthetase complex

Understanding the complex network and multi-functionality of proteins is one of the main objectives of post-genome research. Aminoacyl-tRNA synthetases (ARSs) are the family of enzymes that are essential for cellular protein synthesis and viability that catalyze the attachment of specific amino acids to their cognate tRNAs. However, a lot of evidence has shown that these enzymes are multi-functional proteins that are involved in diverse cellular processes, such as tRNA processing, RNA splicing and trafficking, rRNA synthesis, apoptosis, angiogenesis, and inflammation. In addition, mammalian ARSs form a macromolecular complex with three auxiliary factors or with the elongation factor complex. Although the functional meaning and physiological significance of these complexes are poorly understood, recent data on the molecular interactions among the components for the multi-ARS complex are beginning to provide insights into the structural organization and cellular functions. In this review, the molecular mechanism for the assembly and functional implications of the multi-ARS complex will be discussed.     

Isoform-specific induction of PKC-epsilon by high glucose protects heart-derived H9c2 cells against hypoxic injury

We investigated which PKC isoforms are involved in high glucose-induced protection against hypoxic injury. Treatment for 48 h with high glucose (22 mM) markedly increased the expression of PKC- epsilon in the particulate fraction (213+/-22.1% of the control) but had no effect on other types of PKC isoforms, suggesting that the high glucose-induced increase in PKC expression is isoform-specific. The mRNA level for PKC- epsilon was also substantially increased, reaching its peak after 4h of high glucose treatment. The high glucose increased PKC-epsilon activity in the particulate fraction up to 183+/-32.2% of the control. During hypoxia, the amount of PKC-epsilon in the particulate fraction was remarkably diminished in the low glucose-treated cells, but remained at a higher level in high glucose-treated cells. The treatment with epsilon V1-2 (10 microM), a specific inhibitor of PKC epsilon, abolished the protective effect of high glucose against hypoxia. These results suggest that isoform-specific induction of PKC-epsilon is involved in high glucose-induced protection against hypoxic injury in heart-derived H9c2 cells.