Figure 2. JNK-mediated phosphorylation of T668P regulates APP internalization and processing.

(A) Both Thapsigargin and Rapamycin activate JNK3 in rat hippocampal neurons. At the indicated times, the lysates were harvested and subjected to immunoprecipitation/kinase assays using GST-c-jun as a substrate. As controls, JNK3 Western results are also shown.

(B) Quantification of ³²P-GST-c-jun in (A). The data are represented as means ± SEM (n=2-3).

(C) JNK3 phosphorylates APP at T668P in vitro. Purified GST-APP^ICD proteins were subjected to in vitro kinase assays in the presence of 32P-γ-ATP using active JNK3, p38, CDK5/p35, and GSK-3β according to manufacturer's instruction (Millipore). Only JNK3 and CDK5 phosphorylated APP, while p38 and GSK-3β did not. Note that JNK3 failed to phosphorylate APP, when T668 was mutated to A, but CDK5/p35-dependent phosphorylation still remained. These data suggest that JNK3 phosphorylates T668P, while CDK5/p35 phosphorylates sites other than T668P.

(D) JNK facilitates internalization of APP from the cell surface. To activate the endogenous JNK pathway, hippocampal neurons were treated with 50 ng/ml Anisomycin for the indicated amount of time. After Anisomycin treatment, cells were subjected to cell-surface biotinylation on ice, and the biotinylated APP on the cell-surface was detected following Neutravidin pulldown. Note that the amount of APP on the cell surface was increased initially, which was followed by a decrease with Anisomycin treatment over time. To test whether the changes in APP trafficking were due to JNK activation, a parallel set of cultures were treated with cell-permeable JNK inhibitor, TAT-TI-JIP peptides. Control cultures were treated with cell-permeable control peptides. When JNK activation was inhibited as indicated by the reduction in p-cjun and p-APP^T668 levels, the amount of APP on the cell surface remained the same with Anisomycin treatment over time. These results suggest that JNK activation is necessary for inducing APP internalization.

(E) Quantification of cell-surface biotinylated APP after Anisomycin treatment. Data are represented as means ± SEM. P values were calculated by student t-test.

(F) JNK activation increased APP processing following internalization. The amount of CTF increased with Anisomycin treatment. Following cell-surface biotinylation on ice, neurons were incubated with Anisomycin for 2 hrs at 37°C. At the end of incubation, biotins left on the cell-surface were removed by treating cells with 50 mM DTT before protein extracts were prepared. The internalized APP and its products were subsequently detected by Neutravidin pulldown followed by APP Western blotting as well as 6E10 immunoprecipitation followed by Strepavidin-HRP blotting.

(G) T668P phosphorylation is necessary for APP internalization and processing. Anisomycin treatment reduced the full-length, biotinylated wild type APP from the cell surface, while it increased the amount of biotinylated CTF. On the other hand, Anisomycin failed to induce internalization of and CTF production from the A668P mutant. 293T cells were transfected with the wild type, full-length APP and A668P mutant, and subjected to the same cell surface biotinylation and internalization as with cortical neurons. Also shown are p-JNK and JNK blots in addition to a loading control, ERK blots.

(H) Quantification of the data in G. Data are represented as means ± SEM. P values were calculated by student t-test.