Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide releasing factor-1

RasGRF1 is a member of the Ras guanine nucleotide exchange factor (RasGEF) family of proteins which are directly responsible for the activation of Ras and Rac GTPases. Originally identified as a phosphoprotein, RasGRF1 has been shown to be phosphorylated by protein kinase A and more recently, by the non-receptor tyrosine kinases Ack1 and Src. In this report we show that RasGRF1 interacts with and is phosphorylated by Cdk5 on serine 731 to regulate its steady state levels in mammalian cells as well as in neurons. Phosphorylation on this site by Cdk5 leads to RasGRF1 degradation through a calpain-dependent mechanism. Additionally, cortical neurons from Cdk5 knockout mice have higher levels of RasGRF1 which are reduced when wild-type Cdk5 is transfected into these neurons. In mitotic cells, nuclei become disorganized when RasGRF1 is overexpressed and this is rescued when RasGRF1 is co-expressed with active Cdk5. When RasGRF1 levels are elevated in neurons through overexpression of either the wild-type RasGRF1, or the phosphorylation mutant of RasGRF1 and by the transfection of a dominant negative Cdk5 construct, nuclei appeared condensed and fragmented. On the other hand, a reduction of RasGRF1 levels through p35/Cdk5 overexpression also leads to nuclear condensation in neurons. These data show that phosphorylation of RasGRF1 by Cdk5 tightly regulates its levels, which is essential for proper cellular organization.     

Phosphorylation of p27Kip1 at Thr187 by Cyclin-dependent Kinase 5 Modulates Neural Stem Cell Differentiation

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27^Kip1 at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27^Kip1 siRNA into Cdk5^+/+ NSCs; 2) reduced neuronal differentiation in Cdk5^−/− compared with Cdk5^+/+ NSCs; 3) Cdk5^+/+ NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27^Kip1 (p27/187A) into Cdk5^+/+ NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27^Kip1 at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27^Kip1, a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.     

Role of actin cytoskeleton in LPS-induced NF-kappaB activation and nitric oxide production in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and is known to cause actin cytoskeleton reorganization in a variety of cells including macrophages. Actin cytoskeleton dynamics influence many cell signaling pathways including the NF-kappaB pathway. LPS is also known to induce the expression of many pro-inflammatory genes via the NF-kappaB pathway. Here, we have investigated the role of actin cytoskeleton in LPS-induced NF-kappaB activation and signaling leading to the expression of iNOS and nitric oxide production. Using murine macrophages, we show that disruption of actin cytoskeleton by either cytochalasin D (CytD) or latrunculin B (LanB) does not affect LPS-induced NF-kappaB activation and the expression of iNOS, a NF-kappaB target gene. However, disruption of actin cytoskeleton caused significant reduction in LPS-induced nitric oxide production indicating a role of actin cytoskeleton in the post-translational regulation of iNOS.     

A dosemetric and radiobiological impact of VMAT and 3DCRT on lumbosacral plexuses,an underestimated organ at risk in cervical cancer patients

BackgroundThe purpose of this study was to evaluate dosimetric and radiobiological difference between volumetric modulated arc therapy (VMAT) and 3-dimensional conformal radiotherapy (3DCRT) in organ at risk (OAR) lumbosacral plexus (LSP) in cervical cancer patients.Materials and methods30 patients of cervical cancer who were treated using 3DCRT or VMAT along with chemotherapy followed by brachytherapy were enrolled. LSP was delineated retrospectively. Dosimetric and radiobiological difference was evaluated. Patients were followed for radiation induced lumbosacral plexopathy (RILSP).ResultsMedian follow-up was 12 months (3–16 months). 53.3% of patients were treated by 3DCRT and 46.7% by VMAT. The mean (±SD) LSP volume: 119.03 ± 15 cm³. The mean volume percentages (%) of the LSP: V5, V10, V20, V30, V40, V50, V55, and V60 were 100%, 99.8%, 99.2%, 94.3%, 84.03%, 59.7%, 0%, 0%, respectively. All patients received doses to the LSP in excess of 50 Gy, one patient received 55 Gy. A statistically significant difference was observed in the median value of V20, V30, V40, V50, D50, P2, P4, P7, P8, P9, and P10 across two different techniques of radiotherapy — VMAT and 3DCRT. None of the patients presented with RILSP. NTCP value was less in VMAT plans compared to 3DCRT, which is also statistically significant.ConclusionRILSP is a rare and often refractory complication of pelvic radiotherapy. Advance radiotherapy technique with proper OAR delineation and constraint can prevent the occurrence of RILSP. VMAT has potential benefits for the probability of dose reduction in LSP. Further studies are required focusing on dose distribution in LSP–OAR and radiotherapy modality.     

Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor

We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.     

Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein–protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.     

A nuclear‐localized rice glyoxalase I enzyme,OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni²⁺ or Zn²⁺ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast‐localized GLYI enzyme, OsGLYI‐8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI‐8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI‐8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady‐state kinetics with a low‐affinity and a high‐affinity substrate‐binding component. Loss of AtGLYI‐2, the closest Arabidopsis ortholog of OsGLYI‐8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI‐2 or OsGLYI‐8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus.