NMR analysis of a Tau phosphorylation pattern.
The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimer's diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.
Landrieu
Isabelle
I
CNRS UMR 8525, Institut Pasteur de Lille, 59019 Lille Cedex, France.
Lacosse
Ludovic
L
Leroy
Arnaud
A
Wieruszeski
Jean-Michel
JM
Trivelli
Xavier
X
Sillen
Alain
A
Sibille
Nathalie
N
Schwalbe
Harald
H
Saxena
Krishna
K
Langer
Thomas
T
Lippens
Guy
G
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019311:Staurosporine / Q000737:chemistry / Q000378:metabolism; D016875:tau Proteins / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D016875:tau Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019311:Staurosporine