Oscillating Kinase Activity

Abstract

Protein phosphorylation regulates numerous cellular processes. Although the importance of the spatiotemporal context of second messenger activity has become increasingly apparent, the precise consequences of the spatiotemporal dynamics of second messenger signals on the extent, duration, and specificity of activity of downstream kinases has been less clear. Violin et al. developed C kinase activity reporter (CKAR), a genetically encoded reversible fluorescence resonance energy transfer (FRET)-based reporter of protein kinase C (PKC)-mediated phosphorylation. They used CKAR in combination with novel FRET-based reporters of PKC translocation, phospholipase C (PLC) activity, and diacylglycerol (DAG) to study the regulation of PKC-mediated phosphorylation in MDCK and HeLa cells in the context of second messenger, substrate, kinase, and phosphatase activity and localization. Maximal activation of PKC by phorbol esters did not saturate phosphorylation of cytoplasmic CKAR unless phosphatases were also inhibited; when CKAR was targeted to the plasma membrane, however, phosphatase inhibition had little effect. Moreover, histamine, which caused only a slight increase in FRET of cytosolic CKAR, elicited oscillations of membrane-targeted-CKAR phosphorylation in HeLa cells. These oscillations were synchronized with oscillations in intracellular calcium concentration ([Ca2+]i), were not accompanied by oscillations in DAG or in PLC activity, and correlated with PKC translocation to the plasma membrane. In MDCK cells, however, ATP stimulated synchronous increases in [Ca2+]i, membrane-targeted CKAR phosphorylation, PLC activity, and DAG. These data suggest high fidelity of kinase activity to second messenger dynamics.J. D. Violin, J. Zhang, R. Y. Tsien, A. C. Newton, A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899-909 (2003). [Abstract] [Full Text]