Microheterogeneity of Micro tubule‐Associated τ Proteins Is Due to Differences in Phosphorylation

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Abstract
We have studied the heterogeneity of the microtu-bule-associated τ proteins using τ-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to τ proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sul-fate (SDS)-polyacrylamide gels, with apparent molecular weights between56,000 and66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that >30 isoforms of τ exist. The T proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of τ is not induced by cycling of microtubules, because two-dimensional immunoblots of τ from total brain are almost identical to those of τ from cycled tubules. Adult rat brain τ, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does τ from 7-day-old rats, which appears as only one band on SDS gels. After dephos-phorylation of cow brain τ with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of τ variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with T labeled in vivo with 32PO4 also indicate that the more acidic τ proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of τ proteins exists at all ages and is due, at least in part, to differences in the state of phosphorylation of τ isoforms.