Figure 7.

JNK3 is the principal kinase for APP in vitro and in vivo. a, JNK3 phosphorylates APP more avidly than JNK1 or JNK2. COS7 cells were transfected with APP₆₉₅ and the indicated JNK constructs. The cells were then treated with 10 μm sorbitol (sorb) or vehicle control 30 min before collection. Lysates were probed with a C-terminal-specific anti-APP antibody (C9, left) or a T668 phosphorylation-specific anti-APP antibody (right). b, JNK1 or JNK2 overexpression does not affect the level of AICD. COS7 cells were transfected with APP and either vector JNK1 or JNK2 plasmids, and the resulting lysate was probed for JNK expression (top), APP (middle), and AICD (bottom). V, Vector. c, JNK3 is required for APP phosphorylation in vivo in mouse brain. Protein (30 μg) was loaded in the B6 wild-type (wt) and JNK3^-/- lanes and then detected with polyclonal C9 antibody. d, The absence of JNK1 or JNK2 does not have any effect on APP CTFs. Protein (30 μg) was loaded into each lane of a 10-20% Tris-tricine gel and then probed for APP CTFs. e, Only JNK3 has an effect on AICD levels in mouse brain. Mouse brain lysates were prepared as in c for wild-type, JNK1^-/-, JNK2^-/-, and JNK3^-/- brains. Antibody C9 was used for detection of AICD. f, Steady-state levels of AICD are elevated in JNK3^-/- mouse neurons compared with wild-type control. DIV 4 neurons were prepared and analyzed as in Figure 2, a and b. The amount of full-length APP was slightly lower in the JNK3^-/- neurons; nevertheless, the level of AICD was elevated. This is consistent with the results in total mouse brain lysates. KO, Knock-out; WB, Western blot.