The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains

The differentiation of neurons and the outgrowth of neurites depends on mic rotubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation o f tau23 (the embryonic form of human tau) with mutants in which critical ph osphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repe ats (which regulate the affinity of tau to microtubules) and the SP or TP m otifs in the domains flanking the repeats (which contain epitopes for antib odies diagnostic of Alzheimer's disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that t he phosphorylation pattern of the transfected tau is very similar to that o f neurons, showing that Sf9 cells can be regarded as an approximate model f or the neuronal balance between kinases and phosphatases. We show that muta tions in the repeat domain and in the flanking domains have opposite effect s. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this t ype of phosphorylation accounts for only a minor fraction of the total phos phate. This argues that the temporary detachment of tau from microtubules ( by phosphorylation at KXGS motifs) is a necessary condition for establishin g cell polarity at a critical point in space or time. Conversely, the phosp horylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.