The microtubule-associated protein tau is phosphorylated by Syk

Abstract

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate alpha-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and alpha-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.

Fig. 1

Phosphorylation of tau by Syk in vitro. Recombinant tau 2N4R was expressed in E. coli BL21 (DE3) and purified as described previously . Recombinant human tau (2 μg) was incubated with or without 0.4 μg of Syk or Abl (Invitrogen) in 30 μl of kinase buffer (50 mM Hepes pH 7.4, 5 mM MnCl₂, 5 mM MgCl₂, 0.01% (v/v) Triton X-100, 12 mM beta-glycerophosphate and 1 mM dithiothreithiol) in the presence of 0.2 M ATP for 30 min at 30 °C. 30 μl of SDS-PAGE sample buffer was added to stop the reaction. Control tau was put through the same procedure but without kinase. The phosphorylation reactions were analyzed by Western blotting using 8% gels and P-Tyr-100 (Blot P-Tyr) and tau-5 (Blot Tau). The data presented are representative of three independent experiments.

Fig. 2

Phosphorylation of tau by Syk in cells. (A) CHO cells were co-transfected with 2N4R V5 tagged human tau and constructs expressing Syk-GFP (Tau + Syk-GFP), Abl (Tau + Abl) or empty vector (Tau + pEGFP-N2) as described previously. An additional positive control consisted of tau-transfected cells treated with pervanadate (Tau pervanadate). Cells were harvested after 24 h and tau was immunoprecipitated using anti-V5 antibody. Western blots on the immunoprecipitates were probed with P-Tyr-100 (IP V5, Blot P-Tyr) and with Tau-5 (IP V5, Blot Tau) or Syk N-19 (IP V5, Blot Syk) after membrane stripping. (B) CHO cells were transiently co-transfected with 0N3R untagged human tau and native human Syk (Tau 0N3R + Syk) or transfected with 0N3R tau alone (Tau 0N3R). Cells were harvested after 24 h and tau was immunoprecipitated using tau-5 antibody. Western blots on the immunoprecipitates were probed with P-Tyr-100 (IP Tau, Blot P-Tyr) and Tau-5 (IP Tau, Blot Tau). The data presented are representative of three independent experiments and were confirmed by using another phosphotyrosine antibody (4G10, Upstate, data not shown).

Fig. 3

Tyr-18 is the primary site phosphorylated by Syk in transfected CHO cells. CHO cells were transiently co-transfected with Syk-GFP construct and tau (Tau + Syk-GFP) or Y18F mutant tau (Tau Y18F + Syk-GFP) or Y197F mutant tau (Tau Y197F + Syk-GFP) or Y394F mutant tau (Tau Y394F + Syk-GFP), in which tyrosine 18, 197 or 394 were replaced by phenylalanine. The constructs used were based upon tau 2N4R cDNA and were V5-tagged. Cells were harvested after 24 h and tau was immunoprecipitated using anti-V5 antibody. Western blots of the immunoprecipitates were probed with P-Tyr-100 (IPV5, Blot P-Tyr) and Tau-5 (IPV5, Blot Tau). The data presented are representative of three independent experiments and were confirmed by using another phosphotyrosine antibody (4G10, Upstate, data not shown).

Fig. 4

Stoichiometry and time course of phosphate incorporation into tau following phosphorylation with Syk (solid triangles) or Fyn (open squares). Recombinant human tau (2 μg) was incubated with 30 μl of kinase buffer (as in Fig. 1) in the presence of 0.2 M ATP and 5 μCi [γ-32P]ATP. Ten μl aliquots were pipetted in duplicate onto p81 Whatman papers at the indicated times as described previously . The results are representative of two independent experiments.