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ERK1/2 is an endogenous negative regulator of the -secretase activity

Su-Kyoung Kim^*, Hyun-Jung Park^*,, Hyun Seok Hong^*, Eun Joo Baik, Min Whan Jung and Inhee Mook-Jung^*,1

^* Department of Biochemistry and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; and
Neuroscience program, Ajou University, Suwon, Korea

¹Correspondence: Department of Biochemistry and Cancer Research Institute, Seoul National University College of Medicine, 28 Yungun-dong, Jongro-gu Seoul, 110-799, Korea. E-mail: inhee{at}snu.ac.kr

SPECIFIC AIMS

Although much progress has been made in identifying the components of -secretase complex, which is believed to play an important role in the pathogenesis of Alzheimer’s disease, the endogenous regulatory mechanism of -secretase is currently unknown. This study was undertaken to examine the possibility that MEK1/2-ERK1/2 kinase pathways are involved in regulating -secretase activity.

PRINCIPAL FINDINGS

1. Enhancement of -secretase activity by MEK1/2 inhibitors
We found that -secretase was stimulated by an MEK1/2 inhibitor, but not by a p38 or a JNK inhibitor. Treatment of MEK1/2 specific inhibitors, PD98059 or U0126, dramatically increased -secretase activity in ANPP cells as well as primary cortical cultured cells, as assessed by an in vitro peptide cleavage assay and Western blot that examined the accumulated APP intracellular domain (AICD) fragment and the attenuated APP C-terminal fragments (APP-CTFs). Sandwich ELISA assay showed that inhibition of MEK1/2 increased the levels of both Aß_1–40 and Aß_1–42 peptides from the cultured supernatants. In contrast, a specific p38 kinase inhibitor, SB202190, or a specific JNK inhibitor, SP600125, had no effect on the activity of -secretase, indicating that the stimulating effect is specific to the inhibition of MEK1/2, but not other MAPK pathways. This result shows that -secretase activity can be regulated by MEK1/2 pathway.

2. Enhancement of -secretase activity following ERK1/2 knock-down
Because MEK1/2 activation is known to stimulate ERK1/2, the above results suggest that the -secretase activity is regulated by MEK1/2-ERK1/2 pathway. To confirm the role of ERK1/2 in regulating -secretase activity, siRNA against ERK1/2 was constructed and transfected into ANPP cells. In the ERK1/2 siRNA-transfected cells, -secretase activity was significantly higher than that in positive control siRNA or nonsilencing siRNA (AF488; Alexa Fluor 488) -transfected cells (Fig. 1 A,B). This provides further supporting evidence that -secretase activity is regulated by MEK1/2-ERK1/2 pathway.

View larger version (44K): [in this window] [in a new window]   Figure 1. A, B) ERK1/2 knockdown experiment with RNA interference showed an increase in -secretase activity. C, D) The dominant negative mutant MEK1 cDNA transfected cells increased the -secretase activity. **P < 0.01; ***P < 0.001.

3. Enhancement of -secretase activity in dominant negative mutant MEK1 cDNA transfected cells
To further confirm the role of MEK1/2-ERK1/2 pathway in regulating -secretase activity, the kinase function of MEK1 was blocked by transfecting the cells with the dominant negative mutant MEK1 cDNA (MEK1 D/N). When MEK1 D/N was expressed in ANPP cells, the phosphorylated form of ERK1/2 almost completely disappeared while the total ERK1/2 protein level remained similar compared with the mock-transfected control cells (Fig. 1 C). -Secretase activity in the MEK1 D/N expressed cells was significantly higher than in the mock-transfected cells (Fig. 1 D), further reinforcing the conclusion that MEK1/2-ERK1/2 pathway is involved in regulating -secretase activity.

4. Attenuation of -secretase activity by ERK1/2 and EGF
The above experiments show that the inhibition of MEK1-ERK1/2 increases -secretase activity. Then, conversely, the activation of MEK1-ERK1/2 pathway is expected to lower -secretase activity. This was tested by examining the effects of purified active EKR1 or ERK2 on -secretase activity. To confirm the validity of the assay system, -secretase activity in presenilin (PS) -negative cells was measured as a negative control. As expected, these cells did not have any -secretase activity. On the other hand, ANPP cells showed high levels of -secretase activity, which was inhibited by L685,458 (a -secretase specific inhibitor; positive control). When purified active ERK1 or ERK2 was added to the isolated -secretase complex of the ANPP cell, -secretase activity was dramatically reduced in a dose-dependent manner at 4 h time point. The effects of ERK1 or ERK2 were comparable to that of L685,458. We also examined the effect of EGF, which is a positive stimulator of ERK1/2 signaling pathway, on -secretase activity. The EGF-treated cells had lower levels of -secretase activity compared with the control cells. Combined, these results show that the activation of the ERK1/2 signaling pathway attenuates -secretase activity. When EGF was added to the cells that expressed MEK1 D/N, -secretase activity was enhanced compared with EGF-treated, nontransfected cells. This result corroborates the conclusion that the activation of MEK1-ERK1/2 pathway is negatively correlated with the activity of -secretase.

5. Specific phosphorylation of nicastrin by ERK1/2
Although the above results show that MEK1-ERK1/2 pathway regulates -secretase activity, they do not provide the information on how this is achieved. We first examined the possibility that ERK1/2 regulates expression of -secretase components; There was no change in the level of PS1 N-terminal fragment (NTF)/C-terminal fragment (CTF), nicastrin (NCT), APH-1, or PEN-2 expression in any of the ANPP, CHO-C99, and RGC-5 cells (Fig. 2 A). We then investigated the possibility that ERK1/2 directly interacts with -secretase complex in regulating its activity. Because ERK1/2 is a well-known kinase, the phosphorylation patterns on the -secretase components were examined using [³²P]-ATP in vitro. Surprisingly, only NCT was phosphorylated by active ERK1 or ERK2. PS1 NTF/CTF, APH-1, or PEN-2 was not phosphorylated by active ERK1 or ERK2 (Fig. 2 B).

View larger version (60K): [in this window] [in a new window]   Figure 2. The active ERK1/2 phosphorylated only NCT in the -secretase complex without changing the protein levels of their components. A) Western blot analysis with the -secretase components specific antibodies showed no changes in protein expression with the MEK specific inhibitor treatments in ANPP cells. B) Autoradiogram from the in vitro kinase assay of the purified active ERK1 or ERK2 with [32P-ATP] showed only 32P-labeled NCT even though all 4 components of -secretase were present in this reaction. The phosphorylated form of NCT was increased as the amount of isolated -secretase complex was increased (lane 2<lane 3<lane 4).

Because ERK1/2 phosphorylates NCT in vitro, it is likely that ERK1/2 and NCT bind to each other. Coimmunoprecipitation (coIP) experiments were performed in order to test this possibility. Both the mature and immature forms of NCT bound to both the unphosphorylated and phosphorylated forms of ERK1/2. This suggests that ERK1/2 reduces -secretase activity by phosphorylating NCT. We can then predict that dephosphorylation of -secretase will increase -secretase activity. To test this prediction, phosphatase was added to the isolated -secretase complex of ANPP cell fractions. As predicted, -secretase activity was markedly enhanced following lambda protein phosphatase treatment, similar to a MEK inhibitor treatment or functional blocking of ERK1/2. The enhancing effect of phosphatase was blocked by phosphatase inhibitor cocktail I. These results strongly suggest that ERK1/2 directly phosphorylates NCT of the -secretase complex, causing the down-regulation of -secretase activity in the cells. If ERK1/2 activity is blocked, NCT would be dephosphorylated, leading to the regulation of -secretase activity.

CONCLUSIONS AND SIGNIFICANCE

Although a great deal of progress has been made in identifying the components of -secretase complex, the endogenous regulatory mechanism of -secretase is unknown. This study provides the first evidence that the ERK1/2 is an endogenous regulator of -secretase. On the one hand, when ERK1/2 activity was inhibited, -secretase activity was dramatically enhanced in several different types of cultured cells including primary cultured cortical neurons of the Tg2576 mouse. This was examined in three different ways: by treating the cells with a MEK inhibitor, blocking ERK1/2 expression by small RNA interference,and expressing the dominant negative mutant MEK1. In all three cases, -secretase activity was dramatically enhanced. On the other hand, when ERK1/2 was activated by EGF treatment, -secretase activity was markedly reduced in these cells. Moreover, when purified active ERK1/2 was added to isolated -secretase complex, it attenuated -secretase activity in a dose dependent manner. These results provide unequivocal evidence that ERK1/2 is an endogenous signaling agent that down regulates -secretase activity.

This study also examined which component of -secretase is the potential substrate of ERK1/2 in this process. When purified active ERK1/2 was added to isolated -secretase complex, only NCT was phosphorylated by active ERK1/2 among the four components of -secretase. Moreover, both phosphorylated and unphosphorylated forms of ERK1/2 were found in the -secretase complex and both forms coprecipitated with both the mature and immature forms of NCT. These results demonstrate that ERK1/2 binds to -secretase complex and phosphorylates NCT, thereby inhibiting the activity of -secretase. As the simplest possibility, ERK1/2 might directly interact with NCT to down-regulate -secretase activity. NCT contains several putative serine/threonine phosphorylation sites. A study is under way to examine this possibility; if the possibility proves to be true, mapping the exact phosphorylation sites on NCT may also be possible. We cannot rule out the possibility, however, that ERK1/2 might phosphorylate NCT in an indirect manner by binding to another component of -secretase complex.

It is well known that ERK1/2 mediates the intracellular signaling pathway for cell survival and differentiation. This study suggests a novel role of ERK in reducing the generation of Aß. Because ERK1/2 activation attenuates -secretase activity, it is expected to reduce Aß generation. Therefore, ERK1/2 may play dual roles in activating the cell survival signals and suppressing Aß generation. Hence, ERK1/2 activation might lead to the attenuation of Aß-induced neuronal cell death along with other types of brain damage caused by amyloid plaque, such as glial inflammation. Further studies of the relationship between ERK1/2 and the generation of Aß should improve the understanding of the pathogenesis of AD.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram of relationship between ERK1/2 activation and -secretase activity modulation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4055fje; doi: 10.1096/fj.05-4055fje

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