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Volume 272, Number 7, Issue of February 14, 1997 pp. 4509-4515
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, August 9, 1996, and in revised form, November 25, 1996)

Hanno M. Roder , R. Paul Fracasso , Frederick J. Hoffman , James A. Witowsky , Gary Davis ^¶ and Carla B. Pellegrino ^¶

From the  Institute for Dementia Research and the ^¶ Institute for Research Technologies, Bayer Corporation, West Haven, Connecticut 06516

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 PK40^erk2 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.

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