Table 1.
Acetylated lysines identified in MAP2c
Table 2.
Acetylated lysines identified in MAP4
Fig 1.
MAP-rich fractions and mouse brain lysates contain targets of acetylation.
A) Cortical brain tissue from wild-type (WT) and tau knock-out (KO) mice were high-salt extracted, boiled where indicated, and resulting homogenates were incubated with acetyl-CoA containing reaction buffers. Samples were analyzed by immunoblotting using anti-acetyl-lysine, Ac-K280, and total tau (T49) antibodies. B) MAP-rich fractions from bovine brain were desalted, where indicated, and incubated with CoA or acetyl-CoA followed by immunoblotting similar to (A) above. Shown are representative immunoblot analysis from N = 3 independent experiments.
Fig 2.
Tau, MAP2c, and MAP4 sequence alignments illustrate extensive MTBR homology and conservation of critical lysines
The microtubule-binding repeats 1–4 from tau, MAP2c, and MAP4 were aligned and exact amino acid homology among the MAPs is depicted with red shading. Solid black lines identify two putative MAP acetylation motifs (see Tables 1 and 2 for all identified acetylated residues), and specific acetylated lysines that are enriched within the identified motifs are highlighted above the indicated lysine residues with filled black circles.
Fig 3.
MAP2 and MAP4 are subject to reversible lysine acetylation
A) QBI-293 cells were co-transfected with MAP2c or MAP4-GFP expression plasmids in the presence of CBP to promote full acetylation, followed by immunoprecipitation and immunoblot analysis using acetylated lysine, T46 or GFP antibodies. The monoclonal antibody T46 detects a highly conserved C-terminal epitope that is identical in tau and MAP2c proteins. B) Immunoprecipitation reactions from cells transfected with vector alone (lane 1), MAP2c alone (lane 2), MAP2c and CBP (lane 3), or MAP4 and CBP (lane 4) were performed with T46 or GFP antibodies. Samples were separated by SDS-PAGE and Coomassie stained followed by gel band excision and analysis by mass spectrometry (NanoLC nanospray MS-MS, see methods). In the absence of CBP, no lysine acetylation was detected. In the presence of CBP, MAP2c and MAP4 acetylation sites were identified and full peptide details are listed in Tables 1 and 2. Shown are representative immunoblots from N = 3 independent experiments. Coomassie staining, gel band excision, and mass spectrometry was performed in triplicate confirming the identity of acetylated lysine residues from three independent MAP acetylation experiments.
Fig 4.
MAPs are subject to auto-acetylation within the MTBR domain
A) Recombinant tau-K18 (residues 244–372) or MAP2c (residues 280–388) fragments were incubated in acetylation reactions containing [14C]-labeled acetyl-CoA followed by SDS-PAGE and phosphorimaging using Storm software to detect radiolabeled MAPs. B) Immunoprecipitated 2N4R tau (lane 1), 2N4R-2CA mutant tau (lane 2), or MAP2c proteins (lane 6) derived from QBI-293 lysates were immobilized on agarose beads, incubated with [14C]-Acetyl-CoA, and analyzed by SDS-PAGE and Coomassie staining followed by phosphorimaging analysis. Replicate IgG control samples (lanes 3–5) were used to clearly separate signal intensities from tau and MAP2c sample lanes. C) Purified MTBR fragments from wild-type MAP2c (280–388), a comparable MAP2c fragment containing a cysteine→alanine substitution (C348A), or MAP4 (925–1102) were incubated in acetylation reactions and direct incorporation of radiolabeled acetyl groups was quantified using a liquid scintillation analyzer. Shown are representative analysis using N = 3 technical replicates from N = 3 independent experiments.
Fig 5.
MAP interactions with tubulin impair MAP acetylation
A) Bovine brain derived MAP-rich fractions were incubated in the absence or presence of increasing concentrations of tubulin (1–10 μM), and analyzed in auto-acetylation reactions containing [14C]-acetyl-CoA. B) Reactions similar to (A) above were incubated with unlabeled acetyl-CoA in the presence of tubulin and analyzed by immunoblotting using acetylated lysine (Acetyl-Lys), acetylated tau (Ac-K280), and total tau (T46) antibodies. We note that the addition of tubulin progressively inhibited tau and MAP2 acetylation. Shown are representative gels and immunoblots from N = 3 independent experiments.
Fig 6.
MAP acetylation alters MAP-mediated MT stabilization
QBI-293 cells were transfected with 2N4R-tau (panels a-d), MAP2c (panels e-h), or MAP4 (panels i-l) in the presence of wild-type CBP (top row) or an inactive CBP-LD mutant (bottom row). MT bundling in MAP-transfected cells (green) was determined 48 hr later by acetylated tubulin immunofluorescence (red), an indicator of MAP-induced MT stabilization. Note, all MAPs tested promoted baseline MT bundling in the absence of CBP activity (CBP-LD mutant), which was less elaborated upon MAP acetylation with active wild-type CBP. Scale bar represents 50 μm. m-o) QBI-293 cells were transfected with MAP2c (m) or MAP4 (n) in the presence or absence of wild-type CBP and analyzed by immunoblotting using the indicated antibodies. Shown are representative images and immunoblots from N = 3 independent experiments (immunoblots depict N = 3 technical replicates in the presence of CBP). Quantification of acetylated MTs by densitometry analysis was determined (o). The double asterisk (**) indicates statistical significance with p-value = 0.005, as determined by student t-test.