LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.

The balance between GMD and OFUT1 regulates Notch signaling pathway activity by modulating Notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.

Mutational analysis for enzyme activity of mouse Gal1,3GalNAc 2,3-sialyltransferase (mST3Gal I).

To determine which amino acid residues are essential for the catalytic activity of mouse Gal1,3GalNAc 2,3-sialyltransferase (mST3Gal I), chemical modification and site-directed mutagenesis were employed against tryptophan and cysteine residues located in the predicted catalytic domain. This enzyme was strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues, and the enzyme activity was completely lost at 3 mM, suggesting the involvement of tryptophan residues in the catalytic activity of mST3Gal I. The N-ethylmaleimide, an irreversible reagent for sulfhydryl group, significantly inhibited the enzyme activity. Seven tryptophan and six cysteine residues conserved in the cloned Gal1,3GalNAc 2,3-sialyltransferases were separately substituted into phenylalanine and serine, respectively. The enzymatic activity assay for tryptophan mutants produced in COS cells showed a complete abolishment of the activity in all of the mutants, except that W70F and W97F retained about 60% and 40% activities of wild type, respectively. In the case of cysteine mutants, no enzyme activity was observed like tryptophan mutants, except for C139S. These results suggest that tryptophan and cysteine residues conserved in ST3Gal I are critical for its activity.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A.

The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistance-determining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 Å) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA.

An alternatively spliced form of Met receptor is tumorigenic

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type-Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer.