Regulation of amyloid precursor protein catabolism involves the mitogen-activated protein kinase signal transduction pathway

Abstract

Catabolic processing of the amyloid precursor protein (APP) is subject to regulatory control by protein kinases. We hypothesized that this regulation involves sequential activation of the enzymes mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK). In the present investigation, we provide evidence that MEK is critically involved in regulating APP processing by both nerve growth factor and phorbol esters. Western blot analysis of the soluble N-terminal APP derivative APPs demonstrated that the synthetic MEK inhibitor PD 98059 antagonized nerve growth factor stimulation of both APPs production and ERK activation in PC12 cells. Moreover, PD 98059 inhibited phorbol ester stimulation of APPs production and activation of ERK in both human embryonic kidney cells and cortical neurons. Furthermore, overexpression of a kinase-inactive MEK mutant inhibited phorbol ester stimulation of APP secretion and activation of ERK in human embryonic kidney cell lines. Most important, PD 98059 antagonized phorbol ester-mediated inhibition of Abeta secretion from cells overexpressing human APP695 carrying the "Swedish mutation." Taken together, these data indicate that MEK and ERK may be critically involved in protein kinase C and nerve growth factor regulation of APP processing. The mitogen-activated protein kinase cascade may provide a novel target for altering catabolic processing of APP.

Fig. 1.

PD 98059 inhibits NGF receptor stimulation of APP_s secretion and ERK activation in PC12 cells.A, Top, Densitometric analysis of the effect of NGF (100 ng/ml) on basal APP_s release with or without PD 98059 (10 μm). Data are mean ± SEM of three experiments (*p < 0.05, different from all other treatment groups). Bottom, Representative Western blot of APP_s fragments released in 15 min by PC12 cells alone or in the presence of NGF with or without PD 98059.B, Representative Western blot of phospho-ERK in PC12 cells after a 15 min drug exposure. The increase in immunoreactivity of the phospho-ERK-specific antibody in the presence of NGF was inhibited by PD 98059.

Fig. 2.

PD 98059 antagonizes phorbol ester stimulation of APP_s release in 15 min and ERK activation in HEK 293 cells.A, Top, Densitometric analysis of PMA (0.1 μm) stimulation of APP_s secretion with or without PD 98059 (10 μm). Data are mean ± SEM and represent five experiments for 22C11 (filled columns) or three experiments for WO-2 (hatched columns) (*p < 0.05, different from all other treatment groups). Bottom, Representative Western blot of the effect of PMA on basal APP_s release alone or in the presence of PD 98059. B, Representative Western blot of ERK isoforms with ERK1 C terminus antibody (top) or phospho-ERK forms (bottom) in HEK 293 cells after a 15 min drug exposure. The PMA-induced “electrophoretic shift” was inhibited by PD 98059. Similarly, the increase in phospho-ERK immunoreactivity in the presence of PMA was antagonized by PD 98059.

Fig. 3.

PD 98059 inhibits PKC stimulation of APP_s secretion and ERK activation in cortical neurons.A, Top, Densitometric analysis of PDBu (1 μm) stimulation of APP_s secretion in rat cortical cultures with or without PD 98059 (10 μm). Data are mean ± SEM of five experiments (*p < 0.05, different from all other treatment groups).Bottom, Representative Western blot of the effect of PD 98059 on PDBu stimulation of APP_s release in 15 min.B, Representative Western blot of ERK isoforms with ERK C terminus antibody (top) or phospho-ERK forms (bottom) in cortical cultures after a 1 hr drug exposure: phorbol ester-induced increase in the phosphorylation state of ERK was antagonized by pharmacological inhibition of MEK.

Fig. 4.

The kinase-dead mutant (K97A) inhibits phorbol ester stimulation of APP_s secretion and ERK activation in HEK 293 cells. A, Top, Densitometric analysis of PMA (0.1 μm) stimulation of APP_s secretion in cells expressing the MEK mutant (K97A) or vector alone (Vector). Data are mean ± SEM and represent three experiments for both 22C11 (filled columns) and WO-2 (hatched columns) (*p < 0.05, different from all other treatment groups). Bottom, Representative Western blot of the effect of PMA on basal APP_s release after transient transfection of vector alone or the K97A mutant.B, Top, Representative Western blot of ERK isoforms with an ERK C terminus antibody in HEK 293 cells transfected with the kinase-dead MEK mutant or vector alone. The “electrophoretic shift” induced by PMA treatment in cells expressing vector alone was inhibited in cells expressing the kinase-dead MEK mutant. Middle, Representative Western blot of MEK1 using a rabbit polyclonal antibody raised against the N terminus of MEK1. Bottom, Representative Western blot of cellular APP using a monoclonal antibody generated against the N terminus of APP.

Fig. 5.

PD 98059 antagonizes phorbol ester inhibition of Aβ secretion in K695sw cells. Top, Densitometric analysis of PMA (1 μm) inhibition of Aβ secretion in K695sw cells with or without PD 98059 (10 μm). Data are mean ± SEM of seven experiments [*p < 0.05, different from control (vehicle alone)]. Bottom, Representative Western blot of the effects of the MEK antagonist PD 98059 on PMA inhibition of Aβ release.