PHOSPHORYLATION-DEPENDENT MONOCLONAL TAU ANTIBODIES DO NOT RELIABLY REPORT PHOSPHORYLATION BY EXTRACELLULAR SIGNAL-REGULATED KINASE-2 AT SPECIFIC SITES
Hm. Roder et al., PHOSPHORYLATION-DEPENDENT MONOCLONAL TAU ANTIBODIES DO NOT RELIABLY REPORT PHOSPHORYLATION BY EXTRACELLULAR SIGNAL-REGULATED KINASE-2 AT SPECIFIC SITES, The Journal of biological chemistry, 272(7), 1997, pp. 4509-4515
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).