Abstract
The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.
Highlights
Tau is the principal microtubule-associated protein found in axons in the nervous system [1, 2] where it promotes the nucleation, elongation, stabilization, and bundling of microtubules
Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease
The tyrosine kinases Fyn, cSrc, and Lck have been shown to bind to tau through their SH3 domains [22]
Summary
Plasmids for bacterial expression of human tau, isoforms 0N3R and 2N4R [32], and mutants of 2N4R tau with either one glutamate (S235E) or 18 glutamates in the N-terminal half of the molecule (ETau, here referred to as Tau-E18) [33] were donated by Dr M. Containing 2–10 g of bound recombinant GST fusion protein were incubated at 4 °C with mixing by rotation with 0.2 ml of 10 g/ml tau construct in binding buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.15 M NaCl, 0.5% (v/v) Triton X-100, 2 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 1% (v/v) protease inhibitor mixture For precipitation of GST or GST fusion proteins, lysate supernatants (0.6 ml) were incubated at 0 – 4 °C for 45 min with 5 l of glutathione-Sepharose beads (GE Healthcare) and washed, treated, and analyzed as described above for cosedimenting recombinant proteins Peptides linked at their N termini to biotin via an aminohexanoic acid linker were synthesized by solid-phase procedures, purified chromatographically, characterized by both chromatography and mass spectrophotometry, and dissolved at 1 mg/ml in water. Reference to the SH3 domains from p85␣ and PLC␥1 and from the Src cSrc nSrc nSrc ϩ [22], this work family kinases cSrc and Fgr (results not shown)
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