scholarly journals Phos-tag-based fluorescence resonance energy transfer system for the analysis of protein kinase and phosphatase reactions

2012 ◽  
Vol 56 (Suppl_1) ◽  
pp. s45-s49
Author(s):  
Eiji Kinoshita ◽  
Emiko Kinoshita-Kikuta ◽  
Kei Takiyama ◽  
Masao Somura ◽  
Tohru Koike
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Transfer System ◽  
Fluorescence Resonance

scholarly journals Phospholemman Phosphorylation Alters Its Fluorescence Resonance Energy Transfer with the Na/K-ATPase Pump

2006 ◽  
Vol 281 (43) ◽  
pp. 32765-32773 ◽  
Author(s):  
Julie Bossuyt ◽  
Sanda Despa ◽  
Jody L. Martin ◽  
Donald M. Bers
Keyword(s):  
Protein Kinase C ◽  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Kinase C ◽  
Fluorescence Resonance

Phospholemman (PLM) or FXYD1 is a major cardiac myocyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na/K-pump (NKA) and mediates adrenergic Na/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP/YFP) fusion proteins of Na/K pump and phospholemman and whether phospholemman phosphorylation alters such FRET. Co-expressed NKA-CFP and PLM-YFP in HEK293 cells co-localized in the plasma membrane and exhibited robust FRET. Selective acceptor photobleach increased donor fluorescence (FCFP) by 21.5 ± 4.1% (n = 13), an effect nearly abolished when co-expressing excess phospholemman lacking YFP. Activation of protein kinase C or A progressively and reversibly decreased FRET assessed by either the fluorescence ratio (FYFP/FCFP) or the enhancement of donor fluorescence after acceptor bleach. After protein kinase C activation, forskolin did not further reduce FRET, but after forskolin pretreatment, protein kinase C could still reduce FRET. This agreed with phospholemman phosphorylation measurements: by protein kinase C at both Ser-63 and Ser-68, but by protein kinase A only at Ser-68. Expression of PLM-YFP and PLM-CFP resulted in even stronger FRET than for NKA-PLM (FCFP increased by 37 ± 1% upon YFP photobleach), and this FRET was enhanced by phospholemman phosphorylation, consistent with phospholemman multimerization. Co-expressed PLM-CFP and Na/Ca exchange-YFP were highly membrane co-localized, but FRET was undetectable. We conclude that phospholemman and Na/K-pump are in very close proximity (FRET occurs) and that phospholemman phosphorylation alters the interaction of Na/K-pump and phospholemman.

scholarly journals Stimulus-Specific Distinctions in Spatial and Temporal Dynamics of Stress-Activated Protein Kinase Kinase Kinases Revealed by a Fluorescence Resonance Energy Transfer Biosensor

2009 ◽  
Vol 29 (22) ◽  
pp. 6117-6127 ◽  
Author(s):  
Taichiro Tomida ◽  
Mutsuhiro Takekawa ◽  
Pauline O'Grady ◽  
Haruo Saito
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Cell Population ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A Cell ◽  
Fluorescence Resonance

ABSTRACT The stress-activated protein kinases (SAPKs), namely, p38 and JNK, are members of the mitogen-activated protein kinase family and are important determinants of cell fate when cells are exposed to environmental stresses such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks), which are in turn activated by various SAP2K kinases (SAP3Ks). Because conventional methods, such as immunoblotting using phospho-specific antibodies, measure the average activity of SAP3Ks in a cell population, the intracellular dynamics of SAP3K activity are largely unknown. Here, we developed a reporter of SAP3K activity toward the MKK6 SAP2K, based on fluorescence resonance energy transfer, that can uncover the dynamic behavior of SAP3K activation in cells. Using this reporter, we demonstrated that SAP3K activation occurs either synchronously or asynchronously among a cell population and in different cellular compartments in single cells, depending on the type of stress applied. In particular, SAP3Ks are activated by epidermal growth factor and osmostress on the plasma membrane, by anisomycin and UV in the cytoplasm, and by etoposide in the nucleus. These observations revealed previously unknown heterogeneity in SAPK responses and supplied answers to the question of the cellular location in which various stresses induce stimulus-specific SAPK responses.

Sign in / Sign up

Export Citation Format

Share Document