Imaging Protein Kinase Cα Activation in Cells-Reference-Cited by-同舟云学术

Imaging Protein Kinase Cα Activation in Cells

Published:1999-03-26 Issue:5410 Volume:283 Page:2085-2089
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Ng Tony¹,Squire Anthony²,Hansra Gurdip¹,Bornancin Frederic¹,Prevostel Corinne¹,Hanby Andrew³,Harris William³,Barnes Diana³,Schmidt Sandra²,Mellor Harry¹,Bastiaens Philippe I. H.²,Parker Peter J.¹

Affiliation:

1. Protein Phosphorylation Laboratory and

2. Cell Biophysics Laboratory, Imperial Cancer Research Fund (ICRF), 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.

3. Hedley Atkins Laboratory, Imperial Cancer Research Fund, Guy's Hospital, St. Thomas Street, London, SE1 9RT, UK.

Abstract

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Cα (PKCα) activation is identified and exploited. Activation of PKCα is detected through the binding of fluorescently tagged phosphorylation site–specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCα by FLIM. This approach enabled the imaging of PKCα activation in live and fixed cultured cells and was also applied to pathological samples.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference27 articles.

1. Studies and Perspectives of Protein Kinase C

2. Protein kinase C isoenzymes: divergence in signal transduction?

3. Protein kinase C - a question of specificity

4. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane

5. Thr 250 was identified following in vivo 32 P-labeling of PKCα-transfected COS cells. PKCα was purified from cell extracts fractionated by SDS–polyacrylamide gel electrophoresis and digested with trypsin. Labeled peptides were separated by high-performance liquid chromatography and subjected to Edman degradation to identify the phosphorylation sites. One peptide released 32 P at cycles 1 and 11 predicting the sites threonine-250 (T250) and serine-260 (S260) derived from an incomplete tryptic cleavage. Confirmation of this identification was derived from the generation of phospho-specific T(P)250 and S(P)260 antisera. Both antisera reacted with PKCα confirming occupation of these sites. The S260 site is not discussed further here.

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