Studies on nuclear localization of eNOS in mammalian cells

Abstract

Targeting of eNOS into various sub-cellular locations within the cells plays an important role in human physiology. We have addressed the following questions in this study: a) whether eNOS localization into the nuclei of human cells was a major mode of its regulation or was it only a minor and a specialized mode of regulation that takes place in exceptional cases; b) what were the possible molecular attributes of the eNOS protein that effect its distribution between the nuclear and the cytoplasmic compartments of the cell; and c) what physiological consequences might ensue with a disturbed nuclear distribution of eNOS within the cellular environment. We made an important and a novel observation that eNOS was present in the nuclei of several types of human cells, both normal and cancerous, under conditions of a steady state growth in cell culture and our data clearly suggest that its function in this compartment must be taken into consideration while interpreting its role in the physiology of vascular, cardiac, renal and other systems. The characterization of eNOS protein from the purified nuclei revealed that the nuclear eNOS was largely phosphorylated at the Threonine-495 residue and existed predominantly as a monomer. A major fraction of the nuclear eNOS was associated with nuclear speckles, thought to contain spliceosomes and to be involved in the RNA-metabolism. Association of eNOS with spliceosomes/speckled bodies was specific, because it was found to be physically associated with a splicing factor, SC- 35. We demonstrated that the nuclear eNOS retained its ability to generate NO in vitro when supplemented with the necessary cofactors and substrates. The kinetics of NO-generation from the extracts of purified nuclei showed distinct signatures of enzyme-activation in vitro in a temporal sequence and indicated that the eNOS present in the nuclei may not have been primed to generate optimal amounts of NO.