The abnormal phosphorylation of tau protein on serines and threonines is a hallmark characteristic of the neurofibrillary tangles of Alzheimer's disease (AD). The discovery that tau could be phosphorylated on tyrosine and evidence that A␤ signal transduction involved tyrosine phosphorylation led us to question whether tyrosine phosphorylation of tau occurred during the neurodegenerative process. In this study we determined that human tau tyr18 was phosphorylated by the src family tyrosine kinase fyn. By developing both polyclonal and monoclonal probes specific for phospho-tyr18, we found that the phosphorylation of tau at tyr18 occurred at early developmental stages in mouse but was absent in the adult. Our phosphospecific probes also revealed that paired helical filament preparations exhibited phospho-tyr18 reactivity that was sensitive to phosphotyrosine-specific protein phosphatase treatment. Moreover, immunocytochemical studies indicated that tyrosine phosphorylated tau was present in the neurofibrillary tangles in AD brain. However, the staining pattern excluded neuropil threads and dystrophic neurites indicating that tyrosine phosphorylated tau was distributed in AD brain in a manner dissimilar from other abnormally phosphorylated tau. We also found evidence suggesting that differentially phosphorylated tau existed within degenerating neurons. Our data add new support for a role for fyn in the neurodegenerative process.

The microtubule-associated protein tau interacts with the SH3 domain of non-receptor Src family protein tyrosine kinases. A potential consequence of the SH3 interaction is the upregulation of tyrosine kinase activity. Here we investigated the activation of Src or Fyn by tau, both in vitro and in vivo. Tau increased the kinase activity in in vitro assays and in transfected COS7 cells. In plateletderived growth factor (PDGF)-stimulated fibroblasts, tau appeared to prime Src for activation following PDGF stimulation, as reflected by changes in Src-mediated actin rearrangements. In addition, while fibroblasts normally recovered actin stress fibers by 5-7 hours after PDGF stimulation, tau-expressing cells showed sustained actin breakdown. Microtubule association by tau was not required for the observed changes in actin morphology. Inhibition of Src kinases or a mutant deficient in Src interaction reduced the effects, implicating Src family protein tyrosine kinases as a mediator of the effects of tau on actin rearrangements. Our results provide evidence that the interaction of tau with Src upregulates tyrosine kinase activity and that this interaction allows tau to impact on growth-factor-induced actin remodeling. Journal of Cell Science Tau impacts on actin remodeling Results Tau affects SFK activityWe have previously shown through in vitro binding assays that human tau interacted with the SH3 domain of Fyn and Src (Bhaskar et al., 2005;Lee et al., 1998). Based on the crystal structure of Src, SH3 ligands are thought to 'unclamp' an inactive form of Src, enhancing its activation (Xu et al., 1999;Xu et al., 1997). Our first indication that tau might function in this manner came from experiments initially performed to examine the ability of tyrosine-phosphorylated tau to associate with microtubules. Purified tau and Fyn were incubated in an in vitro kinase reaction to first generate tyrosinephosphorylated tau. The reaction was then added to taxolstabilized microtubules. Upon examining the microtubule pellet for tyrosine-phosphorylated tau, we discovered that the taxol-stabilized tubulin was also tyrosine phosphorylated, presumably by the Fyn that had been carried over from the kinase reaction. Moreover, although Fyn alone was capable of phosphorylating taxol-stabilized microtubules, the presence of tau greatly enhanced the phosphorylation (Fig. 1A, compare lanes 4 and 8).However, because tau binds to microtubules, a possible explanation for the enhanced tubulin phosphorylation was that tau was simply bringing Fyn to the microtubules and facilitating tubulin phosphorylation without affecting Fyn activity. This would predict that a fragment of tau lacking microtubule-binding activity would be incapable of enhancing the phosphorylation. We therefore tested an N-terminal fragment of tau that binds to Fyn SH3 but lacks the microtubule-binding repeats [amino acids 1-184 (Lee et al., 1998)]. By using the same protocol in which the tau fragment was incubated with Fyn and then added to taxol-stabilized microtubules, we found ...

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