Analysis of SHP-1-mediated down-regulation of the TRK-T3 oncoprotein identifies Trk-fused gene (TFG) as a novel SHP-1-interacting protein

SHP-1 is a cytoplasmic SH2 domain containing protein-tyrosine phosphatase (PTP) involved in the negative regulation of multiple signaling pathways in hematopoietic, nervous, and epithelial cells. The thyroid TRK-T3 oncogene consists of the NTRK1 tyrosine kinase domain fused in-frame with sequences of the TFG (TRK-fused gene), encoding a protein of unknown function. TFG contains a coiled-coil domain responsible for TRK-T3 oligomerization. In addition, recent analysis of the sequences outside of the coiled-coil domain suggested possible interactions with other proteins. Based on the presence of a putative SHP-1 SH2-binding site within the TFG sequences, we have investigated the role of the SHP-1 phosphatase in TRK-T3 oncoprotein signaling. In this study we show that SHP-1 interacts with and down-regulates TRK-T3. We provide evidence that SHP-1 SH2 and catalytic domains, respectively, associate with the TFG- and NTRK1-derived portions of TRK-T3. Our data contribute to the definition of cellular mechanisms involved in thyroid tumorigenesis. Moreover, it reveals TFG as a novel protein able to modulate SHP-1 activity.     

Conserved glycines in the C terminus of MinC proteins are implicated in their functionality as cell division inhibitors

Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC(Ng)) and E. coli MinC (MinC(Ec)) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC(Ec) interacts with itself and MinD(Ec); however, no interactions involving MinC(Ng) were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC(Ec) and the C terminus of MinC(Ng), was designed to test for a MinC(Ng)-MinD(Ng) interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC(Ec) G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC(Ec) G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the alpha-7 helix of MinD.     

Differential signalling of purinoceptors in HeLa cells through the extracellular signal-regulated kinase and protein kinase C pathways

We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities.     

Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.     

CpG oligodeoxynucleotides induce Ca2+-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes

Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have been reported to induce antimycobacterial activity both in vitro and in vivo. The present study analyzes the signals leading to CpG ODN-induced antimicrobial activity in monocytes. In this context, CpG, but not GpC, ODN induced cytosolic Ca2+ influx of extracellular origin which, in turn, activated host phospholipase D (PLD). The production of CpG-induced PLD-dependent phosphatidic acid induced the maturation of phagolysosomes and intracellular mycobacterial growth inhibition. These results show the presence of an antimicrobial pathway in monocytes, mediated by Ca2+-dependent PLD which can be useful for the exploitation of novel anti-tuberculosis immunotherapy approaches.     

Fertility in dairy cows following presynchronization and administering twice the luteolytic dose of prostaglandin F2α as one or two injections in the 5-day timed artificial insemination protocol

The objectives were to evaluate pregnancy per AI (P/AI) of dairy cows subjected to the 5-day timed AI protocol under various synchronization and luteolytic treatments. Cows were either presynchronized or received supplemental progesterone during the synchronization protocol, and received a double luteolytic dose of PGF_2α, either as one or two injections. In Experiment 1, dairy cows (n = 737; Holstein = 250, Jersey = 80, and crossbred = 407) in two seasonal grazing dairy farms were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement. The day of AI was considered study Day 0. Half of the cows were presynchronized (G6G: PGF_2α on Day −16 and GnRH on Day −14) and received the 5-day timed AI protocol using 1 mg of cloprostenol, either as a single injection (G6G-S: GnRH on Day −8, PGF_2α on Day −3, and GnRH + AI on Day 0) or divided into two injections of 0.5 mg each (G6G-T: GnRH on Day −8, PGF_2α on Day −3 and −2, and GnRH + AI on Day 0). The remaining cows were not presynchronized and received a controlled internal drug-release (CIDR) insert containing progesterone from GnRH to the first PGF_2α injection of the 5-day timed AI protocol, and 1 mg of cloprostenol either as a single injection on Day -3 (CIDR-S) or divided into two injections of 0.5 mg each on Days -3 and -2 (CIDR-T). Ovaries were examined by ultrasonography on Days −8 and −3 and plasma progesterone concentrations were determined on Days −3 and 0. In Experiment 2, 655 high-producing Holstein cows had their estrous cycle presynchronized with PGF_2α at 46 ± 3 and 60 ± 3 days postpartum and were randomly assigned to receive 50 mg of dinoprost during the 5-day timed AI protocol, either as a single injection or divided into two injections of 25 mg each. Pregnancies per AI were determined on Days 35 and 64 after AI in both experiments. In Experiment 1, presynchronization with G6G increased the proportion of cows with a CL on Day −8 (80.6 vs. 58.8%), ovulation to the first GnRH of the protocol (64.2 vs. 50.2%), and the presence (95.6 vs. 88.4%) and number (1.79 vs. 1.30) of CL at PGF_2α compared with CIDR cows. Luteolysis was greater for two injections compared to a single PGF_2α injection (two PGF_2α = 95.9 vs. single PGF_2α = 72.2%), especially in presynchronized cows (G6G-T = 96.2 vs. G6G-S = 61.7%). For cows not presynchronized, two PGF_2α injections had no effect on P/AI (CIDR-S = 30.2 vs. CIDR-T = 34.3%), whereas for presynchronized cows, it improved P/AI (G6G-S = 28.7 vs. G6G-T = 45.4%). In Experiment 2, the two-PGF_2α injection increased P/AI on Days 35 (two PGF_2α = 44.5 vs. single PGF_2α = 36.4%) and 64 (two PGF_2α = 40.3% vs. single PGF_2α = 32.6%) after AI. Presynchronization and dividing the dose of PGF_2α (either cloprostenol or dinoprost) into two injections increased P/AI in lactating dairy cows subjected to the 5-day timed AI protocol.     

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO

Background

Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.

Methods

Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.

Results

Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.

Conclusion

Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.