Phosphorylation-dependent Monoclonal Tau Antibodies Do Not Reliably Report Phosphorylation by Extracellular Signal-regulated Kinase 2 at Specific Sites

Analysis of phosphorylation of tau, the microtubule-associated proteins hyperphosphorylated in Alzheimer's disease, is often performed using phosphorylation-sensitive monoclonal antibodies thought to report the presence or absence of one or two specific phosphorylations (cognate sites). Using s...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 272; no. 7; pp. 4509 - 4515
Main Authors:	Roder, Hanno M., Fracasso, R. Paul, Hoffman, Frederick J., Witowsky, James A., Davis, Gary, Pellegrino, Carla B.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 14-02-1997 American Society for Biochemistry and Molecular Biology
Subjects:	Antibodies, Monoclonal - metabolism Binding Sites Calcium-Calmodulin-Dependent Protein Kinases - metabolism Electrophoresis, Gel, Two-Dimensional Humans Mitogen-Activated Protein Kinase 1 Peptide Mapping Phosphorus Radioisotopes Phosphorylation Recombinant Proteins - immunology tau Proteins - immunology
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Summary:	Analysis of phosphorylation of tau, the microtubule-associated proteins hyperphosphorylated in Alzheimer's disease, is often performed using phosphorylation-sensitive monoclonal antibodies thought to report the presence or absence of one or two specific phosphorylations (cognate sites). Using several such antibodies we found a much more complicated relationship between phosphorylation at specific sites, as monitored by two-dimensional phosphopeptide mapping, and antibody recognition of these sites. Multiple phosphorylation of tau in several stages by the brain extracellular signal-regulated kinase 2 isoform PK40 suggested that phosphorylation at cognate sites is sometimes necessary (but not sufficient) to induce a change of antibody reactivity and in some cases is not even necessary in the background of multiple phosphorylation at other sites. No single phosphorylation site was found to be responsible for any level of gel mobility shift associated with phosphorylation. Tau acquired its maximal gel mobility retardation and final immunochemical profile at substoichiometric phosphorylation of most sites. This suggests that many alternate phosphorylation patterns can produce the same conformational and immunochemical presentation on sodium dodecyl sulfate-gel electrophoresis. Although PK40erk2 prefers some phosphorylation sites, most notably Ser235, followed by Ser199 or Ser202 and Thr205, the phosphorylation of multiple Ser/Thr-Pro sites is not highly sequential. Ser396 is one of the least preferred sites and seems to require prior phosphorylation at Ser404.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.272.7.4509