Phylogeny and Diversification Patterns among Vesicomyid Bivalves

Vesicomyid bivalves are among the most abundant and diverse symbiotic taxa in chemosynthetic-based ecosystems: more than 100 different vesicomyid species have been described so far. In the present study, we investigated the phylogenetic positioning of recently described vesicomyid species from the Gulf of Guinea and their western Atlantic and Pacific counterparts using mitochondrial DNA sequence data. The maximum-likelihood (ML) tree provided limited support for the recent taxonomic revision of vesicomyids based on morphological criteria; nevertheless, most of the newly sequenced specimens did not cluster with their morphological conspecifics. Moreover, the observed lack of geographic clustering suggests the occurrence of independent radiations followed by worldwide dispersal. Ancestral character state reconstruction showed a significant correlation between the characters “depth” and “habitat” and the reconstructed ML phylogeny suggesting possible recurrent events of ‘stepwise speciation’ from shallow to deep waters in different ocean basins. This is consistent with genus or species bathymetric segregation observed from recent taxonomic studies. Altogether, our results highlight the need for ongoing re-evaluation of the morphological characters used to identify vesicomyid bivalves

Sphingomyelin Synthases Regulate Protein Trafficking and Secretion

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi

Targeting vascular endothelial growth factor receptor 2 and protein kinase d1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-alpha -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways

Phosphorylation At Ser244 By Ck1 Determines Nuclear Localization And Substrate Targeting Of Pkd2

Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells