Figure 1.

Phosphorylation of Aβ at Ser8. (A, B) Primary cultures of mouse cortical neurons were incubated with 10 μM [γ-³²P]ATP in the presence or absence of synthetic Aβ1-40 for 30 min at 37°C. After incubation, Aβ was immunoprecipitated from cell supernatants (A) and cell lysates (B) and was separated by SDS–PAGE, transferred onto nitrocellulose membranes and detected by autoradiography (³²P) and WB. (C) Amino-acid sequence of human Aβ in single letter code. Ser8 (bold) and arginine residue 5 (underlined) resemble a consensus motif for PKA. (D, E) Phosphorylation of Aβ1-40 by primary mouse cortical neurons (1, 1 × 10⁶ cells; 2, 2 × 10⁶ cells) in the absence or presence of 2.5 μM cAMP or 1 μM H-89 were performed as described above and quantified by phospho-imaging (E). ³²P-values represent means±s.d. (n=3). Statistical significance was evaluated by paired t-test (^*P<0.05). (F–H) In vitro phosphorylation of Aβ variants by purified PKA. Synthetic peptides representing Aβ1-16, Aβ17-40, Aβ1-40 (F), Aβ1-40 phosphorylated at Ser8 (pAβ1-40; (G)) and Aβ1-42 (H) were incubated with purified PKA and [γ-³²P]ATP for 15 min at 32°C. pAβ variants were detected by autoradiography followed by WB with appropriate antibodies (Aβ1-16, Aβ1-40, Aβ1-42, pAβ1-40 with antibody 82E1; Aβ17-40 with antibody 4G8). (I, J) Ex vivo phosphorylation in human CSF. Human CSF was incubated with 10 μM [γ-³²P]ATP together with histone (I) or Aβ1-40 (J) in the absence or presence of the indicated kinase inhibitors (H-89, PKI and KT5720) or cAMP. PKA Dep., CSF after immunodepletion of PKA with specific antibodies. The phosphate incorporation was detected by autoradiography (³²P) and quantified by phospho-imaging. Phosphorylation of Aβ was also detected with lower concentrations of ATP (1.0 and 0.1 μM; data not shown). Histone and Aβ was visualized by staining with Ponceau S and WB, respectively. Values represent means±s.d. of three independent experiments.