Participation of the calcium/calmodulin-dependent kinases in hydrogen peroxide-induced Ikappa B phosphorylation in human T lymphocytes.
ABSTRACT NF-kappaB is an important transcription factor that has a role in a variety of responses such as inflammation, oncogenesis, apoptosis, and viral replication. Oxidative stress is well known to induce the activation of NF-kappaB. Cells can be exposed to either endogenously produced oxidants or oxidants produced by surrounding cells. In addition, ischemia reperfusion and certain cancer therapies such as chemotherapy and photodynamic therapy are thought to result in oxygen radical production. Because of the important role that NF-kappaB has in multiple responses, it is critical to determine the mechanisms by which oxidative stress induces NF-kappaB activity. We report that the calmodulin antagonist W-7 and the calcium/calmodulin-dependent (CaM) kinase inhibitors KN-93 and K252a, can block oxidative stress-induced IkappaB phosphorylation in Jurkat T lymphocytes. Furthermore, KN-93 but not KN-92 can block hydrogen peroxide-induced Akt and IKK phosphorylation. In addition, we found that expression of a kinase-dead CaM-KIV construct in two cell lines inhibits IkappaB phosphorylation or degradation and that expression of CaM-KIV augments hydrogen peroxide-induced IkappaB phosphorylation and degradation. Although the CaM kinases appear to be required for this response, increases in intracellular calcium do not appear to be required. These results identify the CaM kinases as potential targets that can be used to minimize NF-kappaB activation in response to oxidative stress.
Article: Inhibition of the CaM-kinases augments cell death in response to oxygen radicals and oxygen radical inducing cancer therapies in MCF-7 human breast cancer cells.[show abstract] [hide abstract]
ABSTRACT: Many cancer treatments induce cell death through lethal oxidative stress. Oxidative stress also induces the activation of the calcium/calmodulin-dependent kinases (CaM-Ks), CaM-KII and CaM-KIV. In turn, the CaM-Ks are known to induce the activation of antiapoptotic signaling pathways, such as Akt, ERK, and NF-kappaB in many different cell types. The aim of this study was to determine the role of CaM-Kinases in resistance to hydrogen peroxide and three oxidative stress-inducing cancer therapies in MCF-7 breast cancer cells. We found that oxidative stress induced CaM-Kinase activity in MCF-7 breast cancer cells and that CaM-K inhibition increased hydrogen peroxide-induced cell death in MCF-7 human breast cancer cells. When MCF-7 cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy in the presence of a CaM-K inhibitor a greater level of cell killing was observed than when cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy alone. In support of this finding, CaM-K inhibition increased hydrogen peroxide-induced apoptosis in MCF-7 cells, as determined by increased number of apoptotic cells, DNA fragmentation, and PARP cleavage. Pharmacological and molecular inhibition indicated that CaM-KII was participating in hydrogen peroxide-induced ERK phosphorylation in breast cancer cells indicating a potential mechanism by which this sensitization occurs. This is the first time that CaM-K inhibition is reported to sensitize cancer cells to reactive oxygen intermediate inducing cancer treatments.Cancer biology & therapy 09/2006; 5(8):1022-30. · 2.64 Impact Factor
Article: Physical association of PDK1 with AKT1 is sufficient for pathway activation independent of membrane localization and phosphatidylinositol 3 kinase.[show abstract] [hide abstract]
ABSTRACT: Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.PLoS ONE 01/2010; 5(3):e9910. · 4.09 Impact Factor
Article: Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS.[show abstract] [hide abstract]
ABSTRACT: Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.PLoS ONE 01/2011; 6(10):e25935. · 4.09 Impact Factor