Characterization of an Intracellular Receptor for Activated Protein Kinase C (RACK) from the MolluscBiomphalaria glabrata, the Intermediate Host forSchistosoma mansoni (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2001
Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMAinduced expression of activated MAPK, suggesting that PKC-, Ras-and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.
International Journal for Parasitology, 2009
For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCb and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCb II . Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78 kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24 h development of miracidia into mother sporocysts; after 31 h and 48 h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31 h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.
Regulatory properties of p105: a novel PKC isoenzyme in mantle tissue from marine mussels
Biochemistry and Cell Biology, 2002
Previous results suggested operative similarities between Apl II from nervous cells of Aplysia californica, ⑀PKC from brain of vertebrates, and p105 from mantle tissue of Mytilus galloprovincialis Lmk., all of them belonging to the nPKC family. The optimal substrate for Apl II and p105 from mussel is protamine sulfate. In contrast, Ca 2+ inhibits p105 but does not affect Apl II. As occurs in other ⑀PKC, p105 is autophosphorylable; however, in Apl II, no P-Tyr residues are detected in the most phosphorylated form. The presence of p105 in all the tissues of M. galloprovincialis studied, proves the important, yet unknown, physiological role that this enzyme must play.
A protein kinase C isozyme is translocated to cytoskeletal elements on activation
Cell regulation, 1990
Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.
Artificial inducers have been used to study signal-transduction pathways involved in metamorphosis of some marine invertebrates. However, the transduction mechanisms for echinoderms have been less explored. In the present study, participation of protein kinase C (PKC), G-protein-coupled receptors (GPCRs), and calcium has been investigated during metamorphosis of the sea urchin Stronglylocentrotus purpuratus. Competent larvae were induced with different drugs that activate (PKC and GP activators, Ca 2ϩ ionophores, and inhibitors of Ca 2ϩ ATPase) or inhibit (PKC, G-protein, and Ca 2ϩ flux inhibitors) metamorphosis. Six of the compounds were effective: the PKC activators TPA and indolactam; the G-protein inhibitors suramin and guanosine; the calcium ionophore A23187, and the calcium ATPase inhibitor thapsigargin. TPA was effective at 0.001 M; indolactam was effective at 0.001 M. In the presence of KCl as inducer, the G-protein inhibitor suramin was effective at 10 M and guanosine at 0.001 M.
Identification of protein kinase C and its potential substrate in Entamoeba histolytica
Comparative biochemistry and physiology. B, Comparative biochemistry, 1990
1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.
Identification of intracellular receptor proteins for activated protein kinase C
Proceedings of the National Academy of Sciences, 1991
Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activationinduced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC.
Fish & Shellfish Immunology, 2006
Mitogen-activated protein kinases (MAPK) from multicellular organisms comprise three subfamilies of enzymes (ERK, p38 and JNK) involved in control of many intracellular processes: cell division, transcription, cell response to different environmental stresses as well as bioactive compounds such as hormones and cytokines . The functional importance of MAPK in mammalian innate immunity is well established [2] but data on the structure and activity of similar molecules in invertebrates remain scarce. The complete nucleotide and amino acid sequences of invertebrate MAPK are available only for several model species including Drosophila melanogaster, Caenorhabditis elegans and Aplysia californica. The latter species is the only one among the Mollusca where MAPK have been studied at the molecular level. Other contemporary reports concerning MAPK activity in molluscan cells are based solely on Western blot techniques using cross-reactive antibodies to corresponding mammalian molecules . Because haemocytes are responsible for the detection and elimination of pathogens in molluscs, this study was initiated to identify MAPK in haemocytes of common periwinkle Littorina littorea (L.) for further analysis on cellular responses triggered in molluscan cells by various stress stimuli. Given the importance of signalling pathways in determining cellular immune responses, it is of considerable interest to investigate how haemocytes react to immune challenge. Knowledge obtained from these experiments could also provide new data on the physiology of molluscan immune cells.