DGKA ELISA Kit

TCR engagement triggers phospholipase C gamma 1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca2+, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase a (DGKa) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKa to the membrane have not been fully elucidated, although Ca2+ influx and tyrosine kinase activation were proposed to be required. We show that DGKa is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKa during TCR activation and shows that pY335DGK alpha is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKa function and suggest a mechanism by which Lck-dependent phosphorylation and Ca2+ elevation regulate DGKa membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKa relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

The goal of this work was to improve the lycopene storage capacity of the E. coli membrane by engineering both morphological and biosynthetic aspects. First, Almgs, a protein from Acholeplasma laidlawii that is involved in membrane bending is overexpressed to expand the storage space for lycopene, which resulted in a 12% increase of specific lycopene production. Second, several genes related to the membrane-synthesis pathway in E. coli, including plsb, plsc, and dgka, were also overexpressed, which led to a further 13% increase. In addition, membrane separation and component analysis confirmed that the increased amount of lycopene was mainly accumulated within the cell membranes. Finally, by integrating both aforementioned modification strategies, a synergistic effect could be observed which caused a 1.32-fold increase of specific lycopene production, from the 27.5 mg/g of the parent to 36.4 mg/g DCW in the engineered strain. This work demonstrates that membrane engineering is a feasible strategy for increasing the production and accumulation of lycopene in E. coli.