A guanosine 5′-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-32P]GTP to ADP to yield (-32P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP

Povećanje letalnog učinka bleomicina na stanice HeLa i V79 s pomoću pčelinjeg otrova

This study investigated possible growth-inhibiting effects of bee venom applied alone or in combination with a cytotoxic drug bleomycin on HeLa and V79 cells in vitro based on clone formation, cell counting, and apoptosis. Melittin, the key component of bee venom, is a potent inhibitor of calmodulin activity, and also a potent inhibitor cell growth and clonogenicity. Intracellular accumulation of melittin correlates with the cytotoxicity of antitumour agents. Previous studies indicated that some calcium antagonists and calmodulin inhibitors enhanced intracellular levels of antitumor agents by inhibiting their outward transport. In this study, treatment of exponentially growing HeLa and V79 cells with bleomycin caused a dose-dependent decrease in cell survival due to DNA damage. This lethal effect was potentiated by adding a non-lethal dose of the bee venom. By preventing repair of damaged DNA, bee venom inhibited recovery from potentially lethal damage induced by bleomycin in V79 and HeLa cells. Apoptosis, necrosis, and lysis were presumed as possible mechanisms by which bee venom inhibited growth and clonogenicity of V79 cells. HeLa cells, on the other hand, showed greater resistance to bee venom. Our findings suggest that bee venom might find a therapeutic use in enhancing cytotoxicity of antitumour agent bleomycin.U uvjetima in vitro istražen je inhibitorni učinak pčelinjeg otrova, samog ili združenog s citostatikom bleomicinom, na rast stanica HeLa i V79. Rabljene su sljedeće metode: brojenje stanica, metoda klonskog rasta i apoptoza. Poznato je da neki antagonisti kalcija i kalmodulinski inhibitori povisuju unutarstaničnu razinu protutumorskih lijekova inhibirajući njihov prijenos iz stanice. Unutarstanična akumulacija melitina izravno povećava citotoksični učinak protutumorskog lijeka. Obrada stanica HeLa i V79 u eksponencijalnoj fazi rasta bleomicinom uzrokuje oštećenje DNA ovisno o dozi te smanjenje broja živih stanica. Uočeno je da se letalni učinak bleomicina može pojačati dodatkom neletalne doze pčelinjeg otrova. Pčelinji otrov pritom inhibira popravak nastalih oštećenja u stanicama HeLa i V79 te sprječava oporavak stanica tretiranih bleomicinom. Apoptoza, nekroza i liza mogući su mehanizmi kojima pčelinji otrov inhibira rast i stvaranje kolonija stanica V79, dok HeLa-stanice pokazuju pojačanu otpornost na pčelinji otrov. Istraživanje također potvrđuje mogućnost uporabe pčelinjeg otrova u povećanju citotoksičnosti bleomicina

Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer

Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer. © 1999 Cancer Research Campaig