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 protein s 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 PR40 suggested that phosphorylation at cognate sites is sometimes necessary (but not sufficient) to induce a change of antibody reactivity and in some case s is not even necessary in the background of multiple phosphorylation at other sites. No single phosphorylation site was found to be respons ible for any level of gel mobility shift associated with phosphorylati on. Tau acquired its maximal gel mobility retardation and final immuno chemical profile at substoichiometric phosphorylation of most sites, T his suggests that many alternate phosphorylation patterns can produce the same conformational and immunochemical presentation on sodium dode cyl sulfate-gel electrophoresis. Although PK40(erk2) prefers some phos phorylation sites, most notably Ser(235), followed by Ser(199) or Ser( 202) and Thr(205), the phosphorylation of multiple Ser/Thr-Pro sites i s not highly sequential. Ser(396) is one of the least preferred sites and seems to require prior phosphorylation at Ser(404).